In vitro evolution in microfluidic systems

ABSTRACT

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalising genetic elements into microcapsules; and (b) sorting the genetic elements which express the gene product having the desired activity; wherein at least one step is under microfluidic control. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.

The present invention relates to methods for use in in vitro evolutionof molecular libraries. In particular, the present invention relates tomethods of selecting nucleic acids encoding gene products in which thenucleic acid and the activity of the encoded gene product are linked bycompartmentation, using microfluidic systems to create and/or handle thecompartments.

Evolution requires the generation of genetic diversity (diversity innucleic acid) followed by the selection of those nucleic acids whichresult in beneficial characteristics. Because the nucleic acid and theactivity of the encoded gene product of an organism are physicallylinked (the nucleic acids being confined within the cells which theyencode) multiple rounds of mutation and selection can result in theprogressive survival of organisms with increasing fitness. Systems forrapid evolution of nucleic acids or proteins in vitro advantageouslymimic this process at the molecular level in that the nucleic acid andthe activity of the encoded gene product are linked and the activity ofthe gene product is selectable.

Recent advances in molecular biology have allowed some molecules to beco-selected according to their properties along with the nucleic acidsthat encode them. The selected nucleic acids can subsequently be clonedfor further analysis or use, or subjected to additional rounds ofmutation and selection.

Common to these methods is the establishment of large libraries ofnucleic acids. Molecules having the desired characteristics (activity)can be isolated through selection regimes that select for the desiredactivity of the encoded gene product, such as a desired biochemical orbiological activity, for example binding activity.

Phage display technology has been highly successful as providing avehicle that allows for the selection of a displayed protein byproviding the essential link between nucleic acid and the activity ofthe encoded gene product (Smith, 1985; Bass et al., 1990; McCafferty etal., 1990; for review see Clackson and Wells, 1994). Filamentous phageparticles act as genetic display packages with proteins on the outsideand the genetic elements which encode them on the inside. The tightlinkage between nucleic acid and the activity of the encoded geneproduct is a result of the assembly of the phage within bacteria. Asindividual bacteria are rarely multiply infected, in most cases all thephage produced from an individual bacterium will carry the same geneticelement and display the same protein.

However, phage display relies upon the creation of nucleic acidlibraries in vivo in bacteria. Thus, the practical limitation on librarysize allowed by phage display technology is of the order of 10⁷ to 10¹¹,even taking advantage of λ phage vectors with excisable filamentousphage replicons. The technique has mainly been applied to selection ofmolecules with binding activity. A small number of proteins withcatalytic activity have also been isolated using this technique,however, selection was not directly for the desired catalytic activity,but either for binding to a transition-state analogue (Widersten andMannervik, 1995) or reaction with a suicide inhibitor (Soumillion etal., 1994; Janda et al., 1997). More recently there have been someexamples of enzymes selected using phage-display by product formation(Atwell & Wells, 1999; Demartis et al., 1999; Jestin et al., 1999;Pederson, et al., 1998), but in all these cases selection was not formultiple turnover.

Specific peptide ligands have been selected for binding to receptors byaffinity selection using large libraries of peptides linked to the Cterminus of the lac repressor LacI (Cull et al, 1992). When expressed inE. coli the repressor protein physically links the ligand to theencoding plasmid by binding to a lac operator sequence on the plasmid.

An entirely in vitro polysome display system has also been reported(Mattheakis et al., 1994; Hanes and Pluckthun, 1997) in which nascentpeptides are physically attached via the ribosome to the RNA whichencodes them. An alternative, entirely in vitro system for linkinggenotype to phenotype by making RNA-peptide fusions (Roberts andSzostak, 1997; Nemoto et al., 1997) has also been described.

However, the scope of the above systems is limited to the selection ofproteins and furthermore does not allow direct selection for activitiesother than binding, for example catalytic or regulatory activity.

In vitro RNA selection and evolution (Ellington and Szostak, 1990),sometimes referred to as SELEX (systematic evolution of ligands byexponential enrichment) (Tuerk and Gold, 1990) allows for selection forboth binding and chemical activity, but only for nucleic acids. Whenselection is for binding, a pool of nucleic acids is incubated withimmobilised substrate. Non-binders are washed away, then the binders arereleased, amplified and the whole process is repeated in iterative stepsto enrich for better binding sequences. This method can also be adaptedto allow isolation of catalytic RNA and DNA (Green and Szostak, 1992;for reviews see Chapman and Szostak, 1994; Joyce, 1994; Gold et al.,1995; Moore, 1995).

However, selection for “catalytic” or binding activity using SELEX isonly possible because the same molecule performs the dual role ofcarrying the genetic information and being the catalyst or bindingmolecule (aptamer). When selection is for “auto-catalysis” the samemolecule must also perform the third role of being a substrate. Sincethe genetic element must play the role of both the substrate and thecatalyst, selection is only possible for single turnover events. Becausethe “catalyst” is in this process itself modified, it is by definitionnot a true catalyst. Additionally, proteins may not be selected usingthe SELEX procedure. The range of catalysts, substrates and reactionswhich can be selected is therefore severely limited.

Those of the above methods that allow for iterative rounds of mutationand selection are mimicking in vitro mechanisms usually ascribed to theprocess of evolution: iterative variation, progressive selection for adesired the activity and replication. However, none of the methods sofar developed have provided molecules of comparable diversity andfunctional efficacy to those that are found naturally. Additionally,there are no man-made “evolution” systems which can evolve both nucleicacids and proteins to effect the full range of biochemical andbiological activities (for example, binding, catalytic and regulatoryactivities) and that can combine several processes leading to a desiredproduct or activity.

There is thus a great need for an in vitro system that overcomes thelimitations discussed above.

In Tawfik and Griffiths (1998), and in International patent applicationPCT/GB98/01889, we describe a system for in vitro evolution thatovercomes many of the limitations described above by usingcompartmentalisation in microcapsules to link genotype and phenotype atthe molecular level.

In Tawfik and Griffiths (1998), and in several embodiments ofInternational patent application WO9902671, the desired activity of agene product results in a modification of the genetic element whichencoded it (and is present in the same microcapsule). The modifiedgenetic element can then be selected in a subsequent step.

Our subsequent international patent application WO0040712 describes avariation of this technology in which the modification of the geneticelement causes a change in the optical properties of the element itself,and which has many advantages over the methods described previously.

The manipulation of fluids to form fluid streams of desiredconfiguration, discontinuous fluid streams, droplets, particles,dispersions, etc., for purposes of fluid delivery, product manufacture,analysis, and the like, is a relatively well-studied art. For example,highly monodisperse gas bubbles, less than 100 microns in diameter, havebeen produced using a technique referred to as capillary flow focusing.In this technique, gas is forced out of a capillary tube into a bath ofliquid, the tube is positioned above a small orifice, and thecontraction flow of the external liquid through this orifice focuses thegas into a thin jet which subsequently breaks into equal-sized bubblesvia a capillary instability. In a related technique, a similararrangement was used to produce liquid droplets in air.

An article entitled “Generation of Steady Liquid Microthreads andMicron-Sized Monodisperse Sprays and Gas Streams,” Phys. Rev. Lett.,80:2, Jan. 12, 1998, 285-288 (Ganan-Calvo) describes formation of amicroscopic liquid thread by a laminar accelerating gas stream, givingrise to a fine spray.

An articled entitled “Dynamic Pattern Formation in a Vesicle-GeneratingMicrofluidic Device,” Phys. Rev. Lett., 86:18, Apr. 30, 2001 (Thorsen,et al.) describes formation of a discontinuous water phase in acontinuous oil phase via microfluidic cross-flow, specifically, byintroducing water, at a “T” junction between two microfluidic channels,into flowing oil.

U.S. Pat. No. 6,120,666, issued Sep. 19, 2000, describes amicofabricated device having a fluid focusing chamber for spatiallyconfining first and second sample fluid streams for analysingmicroscopic particles in a fluid medium, for example in biological fluidanalysis.

U.S. Pat. No. 6,116,516, issued Sep. 12, 2000, describes formation of acapillary microjet, and formation of a monodisperse aerosol viadisassociation of the microjet.

U.S. Pat. No. 6,187,214, issued Feb. 13, 2001, describes atomisedparticles in a size range of from about 1 to about 5 microns, producedby the interaction of two immiscible fluids.

U.S. Pat. No. 6,248,378, issued Jun. 19, 2001, describes production ofparticles for introduction into food using a microjet and a monodisperseaerosol formed when the microjet dissociates.

Microfluidic systems have been described in a variety of contexts,typically in the context of miniaturised laboratory (e.g., clinical)analysis. Other uses have been described as well. For example,International Patent Publication No. WO 01/89789, published Nov. 29,2001 by Anderson, et al., describes multi-level microfluidic systemsthat can be used to provide patterns of materials, such as biologicalmaterials and cells, on surfaces. Other publications describemicrofluidic systems including valves, switches, and other components.

BRIEF DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention, there is provideda method for isolating one or more genetic elements encoding a geneproduct having a desired activity, comprising the steps of:

-   -   (a) compartmentalising the genetic elements into microcapsules;    -   (b) sorting the genetic elements which express gene product (s)        having the desired activity;        wherein at least one step is under microfluidic control.

In the method of the invention, a genetic element may be expressed toform its gene product before or after compartmentalisation; where thegene product is expressed before compartmentalisation, it is linked tothe genetic element such that they are compartmentalised together.

Preferably, at least one step is performed using electronic control offluidic species.

Advantageously at least one step involves fusion or splitting ofmicrocapsules.

Methods for electronic control of fluidic species, as well as splitting(and fusing) of microcapsules under microfluidic control, are describedherein.

Preferably, the method of the invention comprises the steps of:

-   -   (a) compartmentalising the genetic elements into microcapsules;    -   (c) expressing the genetic elements to produce their respective        gene products within the microcapsules; and    -   (d) sorting the genetic elements which encode gene product (s)        having the desired activity.

Alternatively, the method of the invention comprises the steps of:

-   -   (a) expressing the genetic elements to produce their respective        gene products such that the gene products are linked to the        genes encoding them;    -   (b) compartmentalising the genetic elements into microcapsules;        and    -   (c) sorting the genetic elements which encode gene product(s)        having the desired activity.

The microcapsules according to the present invention compartmentalisegenetic elements and gene products such that they remain physicallylinked together.

As used herein, a genetic element is a molecule or molecular constructcomprising a nucleic acid. The genetic elements of the present inventionmay comprise any nucleic acid (for example, DNA, RNA or any analogue,natural or artificial, thereof). The nucleic acid component of thegenetic element may moreover be linked, covalently or non-covalently, toone or more molecules or structures, including proteins, chemicalentities and groups, and solid-phase supports such as beads (includingnonmagnetic, magnetic and paramagnetic beads), and the like. In themethod of the invention, these structures or molecules can be designedto assist in the sorting and/or isolation of the genetic elementencoding a gene product with the desired activity.

Expression, as used herein, is used in its broadest meaning, to signifythat a nucleic acid contained in the genetic element is converted intoits gene product. Thus, where the nucleic acid is DNA, expression refersto the transcription of the DNA into RNA; where this RNA codes forprotein, expression may also refer to the translation of the RNA intoprotein. Where the nucleic acid is RNA, expression may refer to thereplication of this RNA into further RNA copies, the reversetranscription of the RNA into DNA and optionally the transcription ofthis DNA into further RNA molecule(s), as well as optionally thetranslation of any of the RNA species produced into protein. Preferably,therefore, expression is performed by one or more processes selectedfrom the group consisting of transcription, reverse transcription,replication and translation.

Expression of the genetic element may thus be directed into either DNA,RNA or protein, or a nucleic acid or protein containing unnatural basesor amino acids (the gene product) within the microcapsule of theinvention, so that the gene product is confined within the samemicrocapsule as the genetic element.

The genetic element and the gene product thereby encoded are linked byconfining each genetic element and the respective gene product encodedby the genetic element within the same microcapsule. In this way thegene product in one microcapsule cannot cause a change in any othermicrocapsules. In addition, further linking means may be employed tolink gene products to the genetic elements encoding them, as set forthbelow.

The term “microcapsule” is used herein in accordance with the meaningnormally assigned thereto in the art and further described hereinbelow.In essence, however, a microcapsule is an artificial compartment whosedelimiting borders restrict the exchange of the components of themolecular mechanisms described herein which allow the sorting of thegenetic elements according to the function of the gene products whichthey encode.

Preferably, the microcapsules used in the method of the presentinvention will be capable of being produced in very large numbers, andthereby to compartmentalise a library of genetic elements which encodesa repertoire of gene products.

As used herein, a change in optical properties refers to any change inabsorption or emission of electromagnetic radiation, including changesin absorbance, luminescence, phosphorescence or fluorescence. All suchproperties are included in the term “optical”. Microcapsules and/orgenetic elements can be sorted, for example, by luminescence,fluorescence or phosphorescence activated sorting. In a preferredembodiment, flow cytometry is employed to sort microcapsules and/orgenetic elements, for example, light scattering (Kerker, 1983) andfluorescence polarisation (Rolland et al., 1985) can be used to triggerflow sorting. In a highly preferred embodiment genetic elements aresorted using a fluorescence activated cell sorter (FACS) sorter (Norman,1980; Mackenzie and Pinder, 1986). Such a sorting device can beintegrated directly on the microfluidic device, and can use electronicmeans to sort the microcapsules and/or genetic elements. Opticaldetection, also integrated directly on the microfluidic device, can beused to screen the microcapsules and/or genetic elements to trigger thesorting. Other means of control of the microcapsules and/or geneticelements, in addition to charge, can also be incorporated onto themicrofluidic device.

Changes in optical properties may be direct or indirect. Thus, thechange may result in the alteration of an optical property in themicrocapsule or genetic element itself, or may lead indirectly to such achange. For example, modification of a genetic element may alter itsability to bind an optically active ligand, thus indirectly altering itsoptical properties.

Alternatively, imaging techniques can be used to screen thin films ofgenetic elements to allow enrichment for a genetic element withdesirable properties, for example by physical isolation of the regionwhere a genetic element with desirable properties is situated, orablation of non-desired genetic elements. The genetic elements can bedetected by luminescence, phosphorescence or fluorescence.

The sorting of genetic elements may be performed in one of essentiallyseven techniques.

(I) In a first embodiment, the microcapsules are sorted according to anactivity of the gene product or derivative thereof which makes themicrocapsule detectable as a whole. Accordingly, a gene product with thedesired activity induces a change in the microcapsule, or a modificationof one or more molecules within the microcapsule, which enables themicrocapsule containing the gene product and the genetic elementencoding it to be sorted. In this embodiment the microcapsules arephysically sorted from each other according to the activity of the geneproduct(s) expressed from the genetic element(s) contained therein,which makes it possible selectively to enrich for microcapsulescontaining gene products of the desired activity.(II) In a second embodiment, the genetic elements are sorted followingpooling of the microcapsules into one or more common compartments. Inthis embodiment, a gene product having the desired activity modifies thegenetic element which encoded it (and which resides in the samemicrocapsule) in such a way as to make it selectable in a subsequentstep. The reactions are stopped and the microcapsules are then broken sothat all the contents of the individual microcapsules are pooled.Selection for the modified genetic elements enables enrichment of thegenetic elements encoding the gene product(s) having the desiredactivity. Accordingly, the gene product having the desired activitymodifies the genetic element encoding it to enable the isolation of thegenetic element. It is to be understood, of course, that modificationmay be direct, in that it is caused by the direct action of the geneproduct on the genetic element, or indirect, in which a series ofreactions, one or more of which involve the gene product having thedesired activity, leads to modification of the genetic element.(III) In a third embodiment, the genetic elements are sorted followingpooling of the microcapsules into one or more common compartments. Inthis embodiment, a gene with a desired activity induces a change in themicrocapsule containing the gene product and the genetic elementencoding it. This change, when detected, triggers the modification ofthe gene within the compartment. ‘The reactions are stopped and themicrocapsules are then broken so that all the contents of the individualmicrocapsules are pooled. Selection for the modified genetic elementsenables enrichment of the genetic elements encoding the gene product(s)having the desired activity. Accordingly the gene product having thedesired activity induces a change in the compartment which is detectedand triggers the modification of the genetic element within thecompartment so as to allow its isolation. It is to be understood thatthe detected change in the compartment may be caused by the directaction of the gene product, or indirect action, in which a series ofreactions, one or more of which involve the gene product having thedesired activity leads to the detected change.(IV) In a fourth embodiment, the genetic elements may be sorted by amulti-step procedure, which involves at least two steps, for example, inorder to allow the exposure of the genetic elements to conditions whichpermit at least two separate reactions to occur. As will be apparent toa persons skilled in the art, the first microencapsulation step of theinvention must result in conditions which permit the expression of thegenetic elements—be it transcription, transcription and/or translation,replication or the like. Under these conditions, it may not be possibleto select for a particular gene product activity, for example becausethe gene product may not be active under these conditions, or becausethe expression system contains an interfering activity. The methodtherefore comprises expressing the genetic elements to produce theirrespective gene products within the microcapsules, linking the geneproducts to the genetic elements encoding them and isolating thecomplexes thereby formed. This allows for the genetic elements and theirassociated gene products to be isolated from the capsules before sortingaccording to gene product activity takes place. In a preferredembodiment, the complexes are subjected to a furthercompartmentalisation step prior to isolating the genetic elementsencoding a gene product having the desired activity. This furthercompartmentalisation step, which advantageously takes place inmicrocapsules, permits the performance of further reactions, underdifferent conditions, in an environment where the genetic elements andtheir respective gene products are physically linked. Eventual sortingof genetic elements may be performed according to embodiment (I), (II)or (III) above.

Where the selection is for optical changes in the genetic elements, theselection may be performed as follows:

(V) In a fifth embodiment, the genetic elements are sorted followingpooling of the microcapsules into one or more common compartments. Inthis embodiment, a gene product having the desired activity modifies thegenetic element which encoded it (and which resides in the samemicrocapsule) so as to make it selectable as a result of its modifiedoptical properties in a subsequent step. The reactions are stopped andthe microcapsules are then broken so that all the contents of theindividual microcapsules are pooled. The modification of the geneticelement in the microcapsulc may result directly in the modification ofthe optical properties of the genetic element. Alternatively, themodification may allow the genetic elements to be further modifiedoutside the microcapsules so as to induce a change in their opticalproperties. Selection for the genetic elements with modified opticalproperties enables enrichment of the genetic elements encoding the geneproduct(s) having the desired activity. Accordingly, the gene producthaving the desired activity modifies the genetic element encoding it toenable the isolation of the genetic elenrient as a result in a change inthe optical properties of the genetic element. It is to be understood,of course, that modification may be direct, in that it is caused by thedirect action of the gene product on the genetic element, or indirect,in which a series of reactions, one or more of which involve the geneprcsduct having the desired activity, leads to modification of thegenetic element.(VI) In a sixth embodiment, the genetic elements may be sorted by amulti-step procedure, which involves at least two steps, for example, inorder to allow the exposure of the genetic elements to conditions whichpermit at least two separate reactions to occur. As will be apparent topersons skilled in the art, the first microencapsulation step of theinvention advantageously results in conditions which permit theexpression of the genetic elements—be it transcription, transcriptionand/or translation, replication or the like. Under these conditions, itmay not be possible to select for a particular gene product activity,for example because the gene product may not be active under theseconditions, or because the expression system contains an interferingactivity. The method therefore comprises expressing the genetic elementsto produce their respective gene products within the microcapsules,linking the gene products to the genetic elements encoding them andisolating the complexes thereby formed. This allows for the geneticelements and their associated gene products to be isolated from thecapsules before sorting according to gene product activity takes place.In a preferred embodiment, the complexes are subjected to a furthercompartmentalisation step prior to isolating the genetic elementsencoding a gene product having the desired activity. This furthercompartmentalisation step, which advantageously takes place inmicrocapsules, permits the performance of further reactions, underdifferent conditions, in an environment where the genetic elements andtheir respective gene products are physically linked. Eventual sortingof genetic elements may be performed according to embodiment (V) above.

The “secondary encapsulation” may also be performed with geneticelements linked to gene products by other means, such as by phagedisplay, polysome display, RNA-peptide fusion or lac repressor peptidefusion, optionally where expression takes place prior to encapsulation;or even by the encapsulation of whole cells containing the desiredgenetic element.

The selected genetic element(s) may also be subjected to subsequent,possibly more stringent rounds of sorting in iteratively repeated steps,reapplying the method described above either in its entirety or inselected steps only. By tailoring the conditions appropriately, geneticelements encoding gene products having a better optimised activity maybe isolated after each round of selection.

Additionally, the genetic elements isolated after a first round ofsorting may be subjected to mutagenesis before repeating the sorting byiterative repetition of the steps of the method of the invention as setout above. After each round of mutagenesis, some genetic elements willhave been modified in such a way that the activity of the gene productsis enhanced.

Moreover, the selected genetic elements can be cloned into an expressionvector to allow further characterisation of the genetic elements andtheir products.

(VII) In a seventh embodiment, the microcapsules may be sorted usingmicrofiuidic approaches. The microcapsules may be produced usingmicrofluidic droplet formation techniques, such as those describedherein, or by other techniques, for example conventional emulsificationby forcing together two fluid phases. Sorting using microfluidics isapplicable to embodiments I to VI above, and provides enhancedprocessing of microcapsules leading to improved sorting. Microcapsulesmay be split or fused according to methods described herein, or thecontents thereof mixed. Moreover, the contents of the microcapsules maybe analysed and the microcapsules sorted using detectors in microfluidicsystems.

In a second aspect, the invention provides a product when selectedaccording to the first aspect of the invention. As used in this context,a “product” may refer to a gene product, selectable according to theinvention, or the genetic element (or genetic information comprisedtherein).

In a third aspect, the invention provides a method for preparing a geneproduct, the expression of which may result, directly or indirectly, inthe modification the optical properties of a genetic element encodingit, comprising the steps of:

-   -   (a) preparing a genetic element encoding the gene product;    -   (b) compartmentalising genetic elements into microcapsules;    -   (c) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (d) sorting the genetic elements which produce the gene        product(s) having the desired activity using the changed optical        properties of the genetic elements; and    -   (e) expressing the gene product having the desired activity;    -   wherein one or both of steps (b) and (d) is performed under        microfluidic control.

In accordance with the third aspect, step (a) preferably comprisespreparing a repertoire of genetic elements, wherein each genetic elementencodes a potentially differing gene product. Repertoires may begenerated by conventional techniques, such as those employed for thegeneration of libraries intended for selection by methods such as phagedisplay. Gene products having the desired activity may be selected fromthe repertoire, according to the present invention, according to theirability to modify the optical properties of the genetic elements in amanner which differs from that of other gene products. For example,desired gene products may modify the optical properties to a greaterextent than other gene products, or to a lesser extent, including not atall.

In a fourth aspect, the invention provides a method for screening acompound or compounds capable of modulation the activity of a geneproduct, the expression of which may result, directly or indirectly, inthe modification of the optical properties of a genetic element encodingit, comprising the steps of:

-   -   (a) preparing a repertoire of genetic elements encoding gene        product;    -   (b) compartmentalising genetic elements into microcapsules;    -   (c) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (d) sorting the genetic elements which produce the gene        product(s) having the desired activity using the changed optical        properties of the genetic elements; and    -   (e) contacting a gene product having the desired activity with        the compound or compounds and monitoring the modulation of an        activity of the gene product by the compound or compounds;        wherein one or both of steps (b) and (d) is performed under        microfluidic control.

Advantageously, the method further comprises the step of:

-   -   (f) identifying the compound or compounds capable of modulatinig        the activity of the gene product and synthesising said compound        or compounds.

This selection system can be configured to select for RNA, DNA orprotein molecules with catalytic, regulatory or binding activity.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate the splitting of droplets in accordance withone embodiment of the invention;

FIGS. 2A and 2B illustrate an apparatus in accordance with an embodimentof the invention, before the application of an electric field thereto;

FIGS. 3A and 3B illustrate the apparatus of FIGS. 2A and 2B after theapplication of an electric field thereto;

FIGS. 4A and 4B illustrate the apparatus of FIGS. 2A and 2B after theapplication of a reversed electric field thereto;

FIG. 5 is a schematic diagram of droplet splitting, in accordance withone embodiment of the invention;

FIGS. 6A and 6B are schematic diagrams of additional embodiments of theinvention;

FIGS. 7A and 7B are schematic diagrams of the-formation of microfluidicdroplets in accordance with the present invention;

FIGS. 8A-F illustrate the splitting of droplets in accordance with theinvention;

FIGS. 9A-D illustrate the induction of dipoles in droplets in accordancewith the invention;

FIGS. 10A-D illustrate the sorting of microcapsules by altering the flowof carrier fluid in a microfluidic system;

FIGS. 11A-C illustrate the use of pressure changes in the microfluidicsystem to control 10 the direction of flow of droplets;

FIGS. 12A-J illustrate flow patterns for droplets in microfluidicsystems in accordance with the invention;

FIGS. 13A-D illustrate the use of oppositely charged droplets in theinvention;

FIGS. 14A-C are illustrations of the formation and maintenance ofmicrofluidic droplets using three immiscible liquids;

FIGS. 15A-B: Directed evolution of enzymes using microdroplets in amicrofluidic system. FIG. 15A: schematic of the core system. FIG. 15B:process block diagram showing the modules in the core system. Librariesof mutated enzyme genes are encapsulated in aqueous microdroplets (FIG.16A) such that, statistically, the majority of droplets contain no morethan one gene per droplet. Each of these microdroplets is fused with asecond microdroplet (FIG. 16C) containing an in vitro translationsystem. After allowing time for the genes to be translated into proteineach microdroplet is fused with another microdroplet containing aninhibitor of translation (puromycin) and a fluorogenic enzyme substrate.The rate of the enzymatic reaction is determined by measuring thefluorescence of each microdroplet, ideally at multiple points(corresponding to different times). Microdroplets with catalyic ratesover a desired threshold value (e.g. the fastest 1%) will be sorted(FIG. 16D) and collected and the genes contained therein amplified usingthe polymerase chain reaction (PCR). The selected genes will then eitherbe characterised, re-selected directly, or first re-mutated randomly, orrecombined before re-selection.

FIGS. 16A-D: Examples of microdroplet formation and manipulation usingmicrofluidics. FIG. 16A: microdroplets can be created at up to 10⁴ sec¹by hydrodynamic-focussing (top two panels) and show <1.5% polydispersity(bottom panel). FIG. 16B: microdroplets can be split symmetrically orasyminetrically. FIG. 16C: microdroplets carrying positive (+q) andnegative (−q) electrical charges fuse spontaneously. FIG. 16D: chargedmicrodroplets can also be steered using an applied electrical field (E).

FIGS. 17A-F: Charged droplet generation. (FIG. 17A), Oil and waterstreams converge at a 30 micron orifice. A voltage V applied toindium-tin-oxide (ITO) electrodes on the glass produces an electricfield E to capacitively charges the aqueous-oil interface. Drop size isindependent of charge at low field strengths but decreases at higherfields, as shown in the photomicrographs, [(FIG. 17B) V=0, (FIG. 17C)V=400, (FIG. 17D) V=600 and (FIG. 17E) V=800] at higher fields. (FIG.17F) Droplet size as a function of voltage showing the crossover betweenflow-dominated and field-dominated snap-off for three different flowrates of the continuous phase oil (Q_(c),=80 nL/s, 110 nL/s, and 140nL/s). The infusion rate of the water is constant Q_(d)=20 nL/s.}

FIGS. 18A-D: Coalescing drops. (FIG. 18A) Drops having opposite sign ofelectrostatic charge can be generated by applying a voltage across thetwo aqueous streams. (FIG. 18B) In the absence of the field thefrequency and timing of drop formation at the two nozzles areindependent and each nozzle produces a different size drop at adifferent frequency; infusion rates are the same at both nozzles. Afterthe confluence of the two streams, drops from the upper and lowernozzles stay in their respective halves of the stream and due tosurfactant there are no coalescence events even in the case of largeslugs that fill the channel width. (FIG. 18C) With an applied voltage of200V across the 500 micron separation of the nozzles, the dropssimultaneously break-off from the two nozzles and are identical;simultaneous drop formation can be achieved for unequal infusion ratesof the aqueous streams even up to a factor of two difference in volumes.(FIG. 18D) The fraction of the drops that encounter each other andcoalesce increases linearly above a critical field when a surfactant,sorbiton-monooleate 3% is present.

FIGS. 19A-B: Droplets carrying a pH sensitive dye coalesce with dropletsof a different pH fluid. Chaotic advection rapidly mixes the two fluidsthrough a combination of translation and rotation as the droplets passaround corners.

FIGS. 20A-I: Diffusion limited and rapid mixing strategies. (FIG. 20A)Drops meet and coalesce along the direction of E and then move off in aperpendicular direction, as sketched the counter rotating vortices aftercoalescence do not mix the two fluid parts as each vortex contains asingle material. (FIG. 20B) As the drops approach each other theincreasing field causes there interfaces to deform and (FIG. 20C) abridge to jump out connecting the drops, to create (FIG. 20D) in thecase of 20 nm silica particles and MgCl_(—)2 a sharp interface where theparticles begin to gel. (FIG. 20E) A typical unmixed droplet withparticles in one hemisphere. (FIG. 20F) To achieve fast mixing, dropletsare brought together in the direction perpendicular to the electricfield and move off in the direction parallel to the direction theymerged along. Counter rotating vortexes are then created where eachvortex is composed of half of the contentes from each of thepremerger-droplets. (FIG. 20G) Shows a pH sensitive dye in the lowerdrop and a different pH fluid in the upper droplet. (FIG. 20H) Aftermerger the droplets are split by a sharp line. (FIG. 20I) A uniformintensity indicating that mixing has been occurred is achieved in thedroplet after it translates one diameter, typically this takes 1 to 2ms.

FIGS. 21A-B: Time delay reaction module. (FIG. 21A) Droplets ofperfluorodecaline alternate with aqueous droplets in a hexadecanecarrier fluid. The ‘single-file’ ordering of the droplets provides forlong delays with essentially no deviation in the precise spacing ofaqueous droplets or droplet order. (FIG. 21B) Increasing the width andheight of the channel to create a ‘large cross-sectional area’ channelprovides for extremely long time delays from minutes to hours. The exactordering and spacing between the droplets is not maintained in this typeof delay line.

FIGS. 22A-C: Recharging neutral drops. (FIG. 22A) Schematic to rechargeneutral drops by breaking them in the presence of an electric field.Uncharged drops (q=0) are polarized in an electric field (E_(s)≠0), andprovided E_(s) is sufficiently large, as shown in the photomicrograph of(FIG. 22B), they break into two oppositely charged daughter .drops inthe extensional flow at a bifurcation. The enlargement of the dashedrectangle, shown in (FIG. 22C), reveals that the charged drops arestretched in the electric field E_(s) but return to spherical oncontacting the electrodes indicated by dashed vertical lines.

FIG. 23: Detection module. One or more lasers are coupled to an opticalfibre that is used to excite the fluorescence in each droplet as itpasses over the fibre. The fluorescence is collected by the same fibreand dichroic beam splitters separate off specific wavelengths of thefluorescent light and the intensity of the fluorescence is measured witha photomultiplier tube (PMT) after the light passes through a band-passfilter.

FIGS. 24A-D: Manipulating charged drops. In (FIG. 24A) charged dropsalternately enter the right and left channels when there is no fieldapplied (E_(s)=0). The sketch in (FIG. 24B) shows the layout for usingan electric field E_(s) to select the channel charged drops will enterat a bifurcation. When an electric field is applied to the right (FIG.24C), the drops enter the right branch at the bifurcation; they enterthe left branch when the field is reversed (FIG. 24D). After thebifurcation, the distance between drops is reduced to half what it wasbefore indicating the oil stream is evenly divided. The inset of (FIG.24D) shows the deformation in the shape of a highly charged drop in anelectric field.

DEFINITIONS

As used herein, “or” is understood to mean “inclusively or,” i.e., theinclusion of at least one, but including more than one, of a number orlist of elements. In contrast, the term “exclusively or” refers to theinclusion of exactly one element of a number or list of elements.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, should be understood to mean “at leastone.”

The term “about,” as used herein in reference to a numerical parameter(for example, a physical, chemical, electrical, or biological property),will be understood by those of ordinary skill in the art to be anapproximation of a numerical value, the exact value of which may besubject to errors such as those resulting from measurement errors of thenumerical parameter, uncertainties resulting from the variability and/orreproducibility of the numerical parameter (for example, in separateexperiments), and the like.

The term “microcapsule” is used herein in accordance with the meaningnormally assigned thereto in the art and further described hereinbelow.In essence, however, a microcapsule is an artificial compartment whosedelimiting borders restrict the exchange of the components of themolecular mechanisms described herein which allow the identification ofthe molecule with the desired activity. The delimiting borderspreferably completely enclose the contents of the microcapsule.Preferably, the microcapsules used in the method of the presentinvention will be capable of being produced in very large numbers, andthereby to compartmentalise a library of genetic elements. Optionally,the genetic elements can comprise genes attached to microbeads. Themicrocapsules used herein allow mixing and sorting to be performedthereon, in order to facilitate the high throughput potential of themethods of the invention. A microcapsule can be a droplet of one fluidin a different fluid, where the confined components are soluble in thedroplet but not in the carrier fluid. In another embodiment there is athird material defining a wall, such as a membrane.

Arrays of liquid droplets on solid surfaces, multiwell plates and“plugs” in microfluidic systems, that is fluid droplets that are notcompletely surrounded by a second fluid as defined herein, are notmicrocapsules as defined herein.

The term “microbead” is used herein in accordance with the meaningnormally assigned thereto in the art and further described hereinbelow.Microbeads, are also known by those skilled in the art as microspheres,latex particles, beads, or minibeads, are available in diameters from 20nm to 1 mm and can be made from a variety of materials including silicaand a variety of polymers, copolymers and terpolymers. Highly uniformderivatised and non-derivatised nonmagnetic and paramagneticmicroparticles (beads) are commercially available from many sources(e.g. Sigma, Bangs Laboratories, Luminex and Molecular Probes) (Formusekand Vetvicka, 1986).

Microbeads can be “compartmentalised” in accordance with the presentinvention by distribution into microcapsules. For example, in apreferred aspect the microbeads can be placed in a water/oil mixture andemulsified to form a water-in-oil emulsion comprising microcapsulesaccording to the invention. The concentration of the microbeads can beadjusted such that a single microbead, on average, appears in eachmicrocapsule.

As used herein, the “target” is any compound, molecule, orsupramolecular complex. Typical targets include targets of medicalsignificance, including drug targets such as receptors, for example Gprotein coupled receptors and hormone receptors; transcription factors,protein kinases and phosphatases involved in signalling pathways; geneproducts specific to microorganisms, such as components of cell walls,replicases and other enzymes; industrially relevant targets, such asenzymes used in the food industry, reagents intended for research orproduction purposes, and the like.

A “desired activity”, as referred to herein, is the modulation of anyactivity of a target, or an activity of a molecule which is influencedby the target, which is modulatable directly or indirectly by a geneticelement or genetic elements as assayed herein. The activity of thetarget may be any measurable biological or chemical activity, includingbinding activity, an enzymatic activity, an activating or inhibitoryactivity on a third enzyme or other molecule, the ability to causedisease or influence metabolism or other functions, and the like.Activation and inhibition, as referred to herein, denote the increase ordecrease of a desired activity 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold,10 fold, 100 fold or more. Where the modulation is inactivation, theinactivation can be substantially complete inactivation. The desiredactivity may moreover be purely a binding activity, which may or may notinvolve the modulation of the activity of the target bound to.

A compound defined herein as “low molecular weight” or a “smallmolecule” is a molecule commonly referred to in the pharmaceutical artsas a “small molecule”. Such compounds are smaller than polypeptides andother, large molecular complexes and can be easily administered to andassimilated by patients and other subjects. Small molecule drugs canadvantageously be formulated for oral administration or intramuscularinjection. For example, a small molecule may have a molecular weight ofup to 2000 Dalton; preferably up to 1000 Dalton; advantageously between250 and 750 Dalton; and more preferably less than 500 Dalton.

A “selectable change” is any change which can be measured and acted uponto identify or isolate the genetic element which causes it. Theselection may take place at the level of the micro capsule, themicrobead, or the genetic element itself, optionally when complexed withanother reagent. A particularly advantageous embodiment is opticaldetection, in which the selectable change is a change in opticalproperties, which can be detected arid acted upon for instance in a flowsorting device to separate microcapsules or microbeads displaying thedesired change.

As used herein, a change in optical properties refers to any change inabsorption or emission of electromagnetic radiation, including changesin absorbance, luminescence, phosphorescence or fluorescence. All suchproperties are included in the term “optical”. Microcapsules ormicrobeads can be identified and, optionally, sorted, for example, byluminescence, fluorescence or phosphorescence activated sorting. In apreferred embodiment, flow sorting is employed to identify and,optionally, sort microcapsules or microbeads. A variety of opticalproperties can be used for analysis and to trigger sorting, includinglight scattering (Kerker, 1983) and fluorescence polarisation (Rollandet al., 1985).

The genetic elements in microcapsules or on beads can be identifiedusing a variety of techniques familiar to those skilled in the art,including mass spectroscopy, chemical tagging or optical tagging.

As used herein, “microfluidic control” refers to the use of amicrofluidic system comprising microfluidic channels as defined hereinto direct or otherwise control the formation and/or movement ofmicrocapsules (or “droplets”) in order to carry out the methods of thepresent invention. For example, “microfluidic control” of microcapsuleformation refers to the creation of microcapsules using a microfluidicdevice to form “droplets” of fluid within a second fluid, thus creatinga microcapsule. Microcapsules sorted under microfluidic control aresorted, as described herein, using a microfluidic device to perform oneor more of the functions associated with the sorting procedure.“Microfluidic control of fluidic species”, therefore, refers to thehandling of fluids in a microfluidic system as defined in order to carryout the methods of the present invention.

As used herein, a “cell” is given its ordinary meaning as used inbiology. The cell may be any cell or cell type. For example, the cellmay be a bacterium or other single-cell organism, a plant cell, or ananimal cell. If the cell is a single-cell organism, then the cell maybe, for example, a protozoan, a trypanosome, an amoeba, a yeast cell,algae, etc. If the cell is an animal cell, the cell may be, for example,an invertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g.,a zebrafish cell), an amphibian cell (e.g., a frog cell), a reptilecell, a bird cell, or a mammalian cell such as a primate cell, a bovinecell, a horse cell, a porcine cell, a goat cell, a dog cell, a cat cell,or a cell from a rodent such as a rat or a mouse. If the cell is from amulticellular organism, the cell may be from any part of the organism.For instance, if the cell is from an animal, the cell may be a cardiaccell, a fibroblast, a keratinocyte, a heptaocyte, a chondrocyte, aneural cell, a osteocyte, a muscle cell, a blood cell, an endothelialcell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, aneutrophil, a basophil, a mast cell, an eosinophil), a stem cell, etc.In some cases, the cell may be a genetically engineered cell. In certainembodiments, the cell may be a Chinese hamster ovarian (“CHO”) cell or a3T3 cell.

“Microfluidic,” as used herein, refers to a device, apparatus or systemincluding at least one fluid channel having a cross-sectional dimensionof less than 1 mm, and a ratio of length to largest cross-sectionaldimension of at least 3:1. A “microfluidic channel,” as used herein, isa channel meeting these criteria.

The “cross-sectional dimension” of the channel is measured perpendicularto the direction of fluid flow. Most fluid channels in components of theinvention have maximum cross-sectional dimensions less than 2 mm, and insome cases, less than 1 mm. In one set of embodiments, all fluidchannels containing embodiments of the invention are microfluidic orhave a largest cross sectional dimension of no more than 2 mm or 1 mm.In another embodiment, the fluid channels may be formed in part by asingle component (e.g. an etched substrate or moulded unit). Of course,larger channels, tubes, chambers, reservoirs, etc. can be used to storefluids in bulk and to deliver fluids to components of the invention. Inone set of embodiments, the maximum cross-sectional dimension of thechannel(s) containing embodiments of the invention are less than 500microns, less than 200 microns, less than 100 microns, less than 50microns, or less than 25 microns.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs the flow of a fluid. Thechannel can have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore. An open channel generally will include characteristics thatfacilitate control over fluid transport, e.g., structuralcharacteristics (an elongated indentation) and/or physical or chemicalcharacteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

The channel may be of any size, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm or 2 mm, or lessthan about 1 mm, or less than about 500 microns, less than about 200microns, less than about 100 microns, less than about 60 microns, lessthan about 50 microns, less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.In some cases the dimensions of the channel may be chosen such thatfluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art. Insome cases, more than one channel or capillary may be used. For example,two or more channels may be used, where they are positioned inside eachother, positioned adjacent to each other, positioned to intersect witheach other, etc.

As used herein, “integral” means that portions of components are joinedin such a way that they cannot be separated from each other withoutcutting or breaking the components from each other.

A “droplet,” as used herein is an isolated portion of a first fluid thatis completely surrounded by a second fluid. It is to be noted that adroplet is not necessarily spherical, but may assume other shapes aswell, for example, depending on the external environment. In oneembodiment, the droplet has a minimum cross-sectional dimension that issubstantially equal to the largest dimension of the channelperpendicular to fluid flow in which the droplet is located.

The “average diameter” of a population of droplets is the arithmeticaverage of the diameters of the droplets. Those of ordinary skill in theart will be able to determine the average diameter of a population ofdroplets, for example, using laser light scattering or other knowntechniques. The diameter of a droplet, in a non-spherical droplet, isthe mathematically-defined average diameter of the droplet, integratedacross the entire surface. As non-limiting examples, the averagediameter of a droplet may be less than about 1 mm, less than about 500micrometers, less than about 200 micrometers, less than about 100micrometers, less than about 75 micrometers, less than about 50micrometers, less than about 25 micrometers, less than about 10micrometers, or less than about 5 micrometers. The average diameter ofthe droplet may also be at least about 1 micrometer, at least about 2micrometers, at least about 3 micrometers, at least about 5 micrometers,at least about 10 micrometers, at least about 15 micrometers, or atleast about 20 micrometers in certain cases.

As used herein, a “fluid” is given its ordinary meaning, i.e., a liquidor a gas. Preferably, a fluid is a liquid. The fluid may have anysuitable viscosity that permits flow. If two or more fluids are present,each fluid may be independently selected among essentially any fluids(liquids, gases, and the like) by those of ordinary skill in the art, byconsidering the relationship between the fluids. The fluids may each bemiscible or immiscible. For example, two fluids can be selected to beimmiscible within the time frame of formation of a stream of fluids, orwithin the time frame of reaction or interaction. Where the portionsremain liquid for a significant period of time then the fluids should besignificantly immiscible. Where, after contact and/or formation, thedispersed portions are quickly hardened by polymerisation or the like,the fluids need not be as immiscible. Those of ordinary skill in the artcan select suitable miscible or immiscible fluids, using contact anglemeasurements or the like, to carry out the techniques of the invention.

As used herein, a first entity is “surrounded” by a second entity if aclosed loop can be drawn around the first entity through only the secondentity. A first entity is “completely surrounded” if closed loops goingthrough only the second entity can be drawn around the first entityregardless of direction. In one aspect, the first entity may be a cell,for example, a cell suspended in media is surrounded by the media. Inanother aspect, the first entity is a particle. In yet another aspect ofthe invention, the entities can both be fluids. For example, ahydrophilic liquid may be suspended in a hydrophobic liquid, ahydrophobic liquid may be suspended in a hydrophilic liquid, a gasbubble may be suspended in a liquid, etc. Typically, a hydrophobicliquid and a hydrophilic liquid are substantially immiscible withrespect to each other, where the hydrophilic liquid has a greateraffinity to water than does the hydrophobic liquid. Examples ofhydrophilic liquids include, but are not limited to, water and otheraqueous solutions comprising water, such as cell or biological media,ethanol, salt solutions, etc. Examples of hydrophobic liquids include,but are not limited to, oils such as hydrocarbons, silicon oils,fluorocarbon oils, organic solvents etc.

The term “determining,” as used herein, generally refers to the analysisor measurement of a species, for example, quantitatively orqualitatively, or the detection of the presence or absence of thespecies. “Determining” may also refer to the analysis or measurement ofan interaction between two or more species, for example, quantitativelyor qualitatively, or by detecting the presence or absence of theinteraction. Example techniques include, but are not limited to,spectroscopy such as infrared, absorption, fluorescence, UV/visible,FTIR (“Fourier Transform Infrared Spectroscopy”), or Raman; gravimetrictechniques; ellipsometry; piezoelectric measurements; immunoassays;electrochemical measurements; optical measurements such as opticaldensity measurements; circular dichroism; light scattering measurementssuch as quasielectric light scattering; polarimetry; refractometry; orturbidity measurements.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridisation techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc. which are incorporated herein by reference)and chemical methods. In addition Harlow & Lane, A Laboratory ManualCold Spring Harbor, N.Y. is referred to for standard ImmunologicalTechniques.

(A) General Description

The microcapsules of the present invention require appropriate physicalproperties to allow the working of the invention.

First, to ensure that the genetic elements and gene products may notdiffuse between microcapsules, the contents of each microcapsule arepreferably isolated from the contents of the surrounding microcapsules,so that there is no or little exchange of the genetic elements and geneproducts between the microcapsules over the timescale of the experiment.However, the permeability of the microcapsules may be adjusted such thatreagents may be allowed to diffuse into and/or out of the microcapsulesif desired.

Second, the method of the present invention requires that there are onlya limited number of genetic elements per microcapsule. This ensures thatthe gene product of an individual genetic element will be isolated fromother genetic elements. Thus, coupling between genetic element and geneproduct will be highly specific. The enrichment factor is greatest withon average one or fewer genetic elements per microcapsule, the linkagebetween nucleic acid and the activity of the encoded gene product beingas tight as is possible, since the gene product of an individual geneticelement will be isolated from the products of all other geneticelements. However, even if the theoretically optimal situation of, onaverage, a single genetic element or less per microcapsule is not used,a ratio of 5, 10, 50, 100 or 1000 or more genetic elements permicrocapsule may prove beneficial in sorting a large library. Subsequentrounds of sorting, including renewed encapsulation with differinggenetic element distribution, will permit more stringent sorting of thegenetic elements. Preferably, there is a single genetic element, orfewer, per microcapsule.

Third, the formation and the composition of the microcapsulesadvantageously does not abolish the function of the machinery theexpression of the genetic elements and the activity of the geneproducts.

Consequently, any microencapsulation system used preferably fulfilsthese three requirements. The appropriate system(s) may vary dependingon the precise nature of the requirements in each application of theinvention, as will be apparent to the skilled person.

A wide variety of microencapsulation procedures are available (seeBenita, 1996) and may be used to create the microcapsules used inaccordance with the present invention.

Indeed, more than 200 microencapsulation methods have been identified inthe literature (Finch, 1993).

Enzyme-catalysed biochemical reactions have also been demonstrated inmicrocapsules generated by a variety of other methods. Many enzymes areactive in reverse micellar solutions (Bru & Walde, 1991; Bru & Walde,1993; Creagh et al., 1993; Haber et al., 1993; Kumar et al., 1989; Luisi& B., 1987; Mao & Walde, 1991; Mao et al., 1992; Perez et al., 1992;Walde et al., 1994; Walde et al., 1993; Walde et al., 1988) such as theAOT-isooctane-water system (Menger & Yamada, 1979).

Microcapsules can also be generated by interfacial polymerisation andinterfacial complexation (Whateley, 1996). Microcapsules of this sortcan have rigid, nonpermeable membranes, or semipermeable membranes.Semipermeable microcapsules bordered by cellulose nitrate membranes,polyamide membranes and lipid-polyamide membranes can all supportbiochemical reactions, including multienzyme systems (Chang, 1987;Chang, 1992; Lim, 1984). Alginate/polylysine microcapsules (Lim & Sun,1980), which can be formed under very mild conditions, have also provento be very biocompatible, providing, for example, an effective method ofencapsulating living cells and tissues (Chang, 1992; Sun et al., 1992).

Non-membranous microencapsulation systems based on phase partitioning ofan aqueous environment in a colloidal system, such as an emulsion, mayalso be used.

Preferably, the microcapsules of the present invention are formed fromemulsions; heterogeneous systems of two immiscible liquid phases withone of the phases dispersed in the other as droplets of microscopic orcolloidal size (Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant,1984).

Emulsions may be produced from any suitable combination of immiscibleliquids. Preferably the emulsion of the present invention has “water”(an aqueous liquid containing the biochemical components) as the phasepresent in the form of finely divided droplets (the disperse, internalor discontinuous phase) and a hydrophobic, immiscible liquid (an ‘oil’)as the matrix in which these droplets are suspended (the nondisperse,continuous or external phase). Such emulsions are termed ‘water-in-oil’(W/O). This has the advantage that the entire aqueous phase containingthe biochemical components is compartmentalised in discreet droplets(the internal phase). The external phase, being a hydrophobic liquid,generally contains none of the biochemical components and hence isinert.

The emulsion may be stabilised by addition of one or more surface-activeagents (surfactants). These surfactants are termed emulsifying agentsand act at the water/oil interface to prevent (or at least delay)separation of the phases. Many oils and many emulsifiers can be used forthe generation of water-in-oil emulsions; a recent compilation listedover 16,000 surfactants, many of which are used as emulsifying agents(Ash and Ash, 1993). Suitable oils include light white mineral oil anddecane. Suitable surfactants include: non-ionic surfactants (Schick,1966) such as sorbitan monooleate (Span™ 80; ICI), sorbitan monosteatate(Span™ 60; ICI), polyoxyethylenesorbitan monooleate (Tween™ 80; ICI),and octylphenoxyethoxyethanol (Triton X-100); ionic surfactants such assodium cholate and sodium taurocholate and sodium deoxycholate;chemically inert silicone-based surfactants such aspolysiloxane-polycetyl-polyethylene glycol copolymer (Cetyl DimethiconeCopolyol) (e.g. Abi™ EM90; Goldschmidt); and cholesterol.

Emulsions with a fluorocarbon (or perfluorocarbon) continuous phase(Krafft et al., 2003; Riess, 2002) may be particularly advantageous. Forexample, stable water-in-perfluorooctyl bromide andwater-in-perfluorooctylethane emulsions can be formed using F-alkyldimorpholinophosphates as surfactants (Sadtler et al., 1996).Non-fluorinated compounds are essentially insoluble in fluorocarbons andperfluorocarbons (Curran, 1998; Hildebrand and Cochran, 1949; Hudlicky,1992; Scott, 1948; Studer et al., 1997) and small drug-like molecules(typically <500 Da and Log P<5) (Lipinski et al., 2001) arecompartmentalised very effectively in the aqueous microcapsules ofwater-in-fluorocarbon and water-in-perfluorocarbon emulsions—with littleor no exchange between microcapsules.

Creation of an emulsion generally requires the application of mechanicalenergy to force the phases together. There are a variety of ways ofdoing this which utilise a variety of mechanical devices, includingstirrers (such as magnetic stir-bars, propeller and turbine stirrers,paddle devices and whisks), homogenisers (including rotor-statorhomogenisers, high-pressure valve homogenisers and jet homogenisers),colloid mills, ultrasound and ‘membrane emulsification’ devices (Becher,1957; Dickinson, 1994), and microfluidic devices (Umbanhowar et al.,2000).

Complicated biochemical processes, notably gene transcription andtranslation are also active in aqueous microcapsules formed inwater-in-oil emulsions. This has enabled compartmentalisation inwater-in-oil emulsions to be used for the selection of genes, which aretranscribed and translated in emulsion microcapsules and selected by thebinding or catalytic activities of the proteins they encode (Doi andYanagawa, 1999; Griffiths and Tawfik, 2003; Lee et al., 2002; Sepp etal., 2002; Tawfik and Griffiths, 1998). This was possible because theaqueous microcapsules formed in the emulsion were generally stable withlittle if any exchange of nucleic acids, proteins, or the products ofenzyme catalysed reactions between microcapsules.

The technology exists to create emulsions with volumes all the way up toindustrial scales of thousands of litres (Becher, 1957; Sherman, 1968;Lissant, 1974; Lissant, 1984).

The preferred microcapsule size will vary depending upon the preciserequirements of any individual selection process that is to be performedaccording to the present invention. In all cases, there will be anoptimal balance between gene library size, the required enrichment andthe required concentration of components in the individual microcapsulesto achieve efficient expression and reactivity of the gene products.

The processes of expression occurs within each individual microcapsuleprovided by the present invention. Both in vitro transcription andcoupled transcription-translation become less efficient at sub-nanomolarDNA concentrations. Because of the requirement for only a limited numberof DNA molecules to be present in each microcapsule, this therefore setsa practical upper limit on the possible microcapsule size. Preferably,the mean volume of the microcapsules is less that 5.2×10⁻¹⁶ m³,(corresponding to a spherical microcapsule of diameter less than 10 μm,more preferably less than 6.5×10⁻¹⁷ m³ (5 μm diameter), more preferablyabout 4.2×10⁻¹⁸ m³ (2 μm diameter) and ideally about 9×10⁻¹⁸ m³ (2.6 μmdiameter).

The effective DNA or RNA concentration in the microcapsules may beartificially increased by various methods that will be well-known tothose versed in the art. These include, for example, the addition ofvolume excluding chemicals such as polyethylene glycols (PEG) and avariety of gene amplification techniques, including transcription usingRNA polymerases including those from bacteria such as E. coli (Roberts,1969; Blattner and Dahlberg, 1972; Roberts et al., 1975; Rosenberg etal., 1975), eukaryotes e.g. (Weil et al., 1979; Manley et al, 1983) andbacteriophage such as T7, T3 and SP6 (Melton et al., 1984); thepolymerase chain reaction (PCR) (Saiki et al., 1988); Qb replicaseamplification (Miele et al., 1983; Cahill et al., 1991; Chetverin andSpirin, 1995; Katanaev et al., 1995); the ligase chain reaction (LCR)(Landegren et al, 1988; Barany, 1991); and self-sustained sequencereplication system (Fahy et al., 1991) and strand displacementamplification (Walker et al, 1992). Gene amplification techniquesrequiring thermal cycling such as PCR and LCR may be used if theemulsions and the in vitro transcription or coupledtranscription-translation systems are thermostable (for example, thecoupled transcription-translation systems can be made from athermostable organism such as Thermus aquaticus).

Increasing the effective local nucleic acid concentration enables largermicrocapsules to be used effectively. This allows a preferred practicalupper limit to the microcapsule volume of about 5.2×10⁻¹⁶ m³(corresponding to a sphere of diameter 10 μm).

The microcapsule size is preferably sufficiently large to accommodateall of the required components of the biochemical reactions that areneeded to occur within the microcapsule. For example, in vitro, bothtranscription reactions and coupled transcription-translation reactionsrequire a total nucleoside triphosphate concentration of about 2 mM.

For example, in order to transcribe a gene to a single short RNAmolecule of 500 bases in length, this would require a minimum of 500molecules of nucleoside triphosphate per microcapsule (8.33×10⁻²²moles). In order to constitute a 2 mM solution, this number of moleculesis contained within a microcapsule of volume 4.17×10⁻¹⁹ litres(4.17×10⁻²² m³ which if spherical would have a diameter of 93 nm.

Furthermore, particularly in the case of reactions involvingtranslation, it is to be noted that the ribosomes necessary for thetranslation to occur are themselves approximately 20 nm in diameter.Hence, the preferred lower limit for microcapsules is a diameter ofapproximately 0.1 μm (100 nm).

Therefore, the microcapsule volume is preferably of the order of between5.2×10⁻²² m³ and 5.2×10⁻¹⁶ m³ corresponding to a sphere of diameterbetween 0.1 μm and 10 μm, more preferably of between about 5.2×10⁻¹⁹ m³and 6.5×10⁻¹⁷ m³ (1 μm and 5 μm). Sphere diameters of about 2.6 μm aremost advantageous.

It is no coincidence that the preferred dimensions of the compartments(droplets of 2.6 μm mean diameter) closely resemble those of bacteria,for example, Escherichia are 1.1−1.5×2.0-6.0 μm rods and Azotobacter are1.5-2.0 1. μm diameter ovoid cells. In its simplest form, Darwinianevolution is based on a ‘one genotype one phenotype’ mechanism. Theconcentration of a single compartmentalised gene, or genome, drops from0.4 nM in a compartment of 2 μm diameter, to 25 pM in a compartment of 5μm diameter. The prokaryotic transcription/translation machinery hasevolved to operate in compartments of ˜1-2 μm diameter, where singlegenes are at approximately nanomolar concentrations. A single gene, in acompartment of 2.6 μm diameter is at a concentration of 0.2 nM. Thisgene concentration is high enough for efficient translation.Compartmentalisation in such a volume also ensures that even if only asingle molecule of the gene product is formed it is present at about 0.2nM, which is important if the gene product is to have a modifyingactivity of the genetic element itself. The volume of the microcapsuleis thus selected bearing in mind not only the requirements fortranscription and translation of the genetic element, but also themodifying activity required of the gene product in the method of theinvention.

The size of emulsion microcapsules may be varied simply by tailoring theemulsion conditions used to form the emulsion according to requirementsof the selection system. The larger the microcapsule size, the larger isthe volume that will be required to encapsulate a given genetic elementlibrary, since the ultimately limiting factor will be the size of themicrocapsule and thus the number of microcapsules possible per unitvolume.

The size of the microcapsules is selected not only having regard to therequirements.of the transcription/translation system, but also those ofthe selection system employed for the genetic element. Thus, thecomponents of the selection system, such as a chemical modificationsystem, may require reaction volumes and/or reagent concentrations whichare not optimal for transcription/translation. As set forth herein, suchrequirements may be accommodated by a secondary re-encapsulation step;moreover, they may be accommodated by selecting the microcapsule size inorder to maximise transcription/translation and selection as a whole.Empirical determination of optimal microcapsule volume and reagentconcentration, for example as set forth herein, is preferred.

A “genetic element” in accordance with the present invention is asdescribed above. Preferably, a genetic element is a molecule orconstruct selected from the group consisting of a DNA molecule, an RNAmolecule, a partially or wholly artificial nucleic acid moleculeconsisting of exclusively synthetic or a mixture of naturally-occurringand synthetic bases, any one of the foregoing linked to a polypeptide,and any one of the foregoing linked to any other molecular group orconstruct. Advantageously, the other molecular group or construct may beselected from the group consisting of nucleic acids, polymericsubstances, particularly beads, for example polystyrene beads, andmagnetic or paramagnetic substances such as magnetic or paramagneticbeads.

The nucleic acid portion of the genetic element may comprise suitableregulatory sequences, such as those required for efficient expression ofthe gene product, for example promoters, enhancers, translationalinitiation sequences, polyadenylation sequences, splice sites and thelike.

As will be apparent from the following, in many cases the polypeptide orother molecular group or construct is a ligand or a substrate whichdirectly or indirectly binds to or reacts with the gene product in orderto alter the optical properties of the genetic element. This allows thesorting of the genetic element on the basis of the activity of the geneproduct. The ligand or substrate can be connected to the nucleic acid bya variety of means that will be apparent to those skilled in the art(see, for example, Hermanson, 1996).

One way in which the nucleic acid molecule may be linked to a ligand orsubstrate is through biotinylation. This can be done by PCRamplification with a 5′-biotinylation primer such that the biotin andnucleic acid are covalently linked.

The ligand or substrate can be attached to the modified nucleic acid bya variety of means that will be apparent to those of skill in the art(see, for example, Hermanson, 1996). A biotinylated nucleic acid may becoupled to a polystyrene or paramagnetic microbead (0.02 to approx. 5.0μm in diameter) that is coated with avidin or streptavidin, that willtherefore bind the nucleic acid with very high affinity. This bead canbe derivatised with substrate or ligand by any suitable method such asby adding biotinylated substrate or by covalent coupling.

Alternatively, a biotinylated nucleic acid may be coupled to avidin orstreptavidin complexed to a large protein molecule such as thyroglobulin(669 Kd) or ferritin (440 Kd). This complex can be derivatised withsubstrate or ligand, for example by covalent coupling to the E-aminogroup of lysines or through a non-covalent interaction such asbiotin-avidin.

The substrate may be present in a form unlinked to the genetic elementbut containing an inactive “tag” that requires a further step toactivate it such as photoactivation (e.g. of a “caged” biotin analogue,(Sundberg et al., 1995; Pirrung and Huang, 1996)). The catalyst to beselected then converts the substrate to product. The “tag” is thenactivated and the “tagged” substrate and/or product bound by atag-binding molecule (e.g. avidin or streptavidin) complexed with thenucleic acid. The ratio of substrate to product attached to the nucleicacid via the “tag” will therefore reflect the ratio of the substrate andproduct in solution.

An alternative is to couple the nucleic acid to a product-specificantibody (or other product-specific molecule). In this scenario, thesubstrate (or one of the substrates) is present in each microcapsuleunlinked to the genetic element, but has a molecular “tag” (for examplebiotin, DIG or DNP or a fluorescent group). When the catalyst to beselected converts the substrate to product, the product retains the“tag” and is then captured in the microcapsule by the product-specificantibody. In this way the genetic element only becomes associated withthe “tag” when it encodes or produces an enzyme capable of convertingsubstrate to product.

The terms “isolating”, “sorting” and “selecting”, as well as variationsthereof; are used herein. Isolation, according to the present invention,refers to the process of separating an entity from a heterogeneouspopulation, for example a mixture, such that it is free of at least onesubstance with which it was associated before the isolation process. Ina preferred embodiment, isolation refers to purification of an entityessentially to homogeneity. Sorting of an entity refers to the processof preferentially isolating desired entities over undesired entities. Inas far as this relates to isolation of the desired entities, the terms“isolating” and “sorting” are equivalent. The method of the presentinvention permits the sorting of desired genetic elements from pools(libraries or repertoires) of genetic elements which contain the desiredgenetic element. Selecting is used to refer to the process (includingthe sorting process) of isolating an entity according to a particularproperty thereof.

In a highly preferred application, the method of the present inventionis useful for sorting libraries of genetic elements. The inventionaccordingly provides a method according to preceding aspects of theinvention, wherein the genetic elements are isolated from a library ofgenetic elements encoding a repertoire of gene products. Herein, theterms “library”, “repertoire” and “pool” are used according to theirordinary signification in the art, such that a library of geneticelements encodes a repertoire of gene products. In general, librariesare constructed from pools of genetic elements arid have propertieswhich facilitate sorting.

Initial selection of a genetic element from a genetic element libraryusing the present invention will in most cases require the screening ofa large number of variant genetic elements. Libraries of geneticelements can be created in a variety of different ways, including thefollowing.

Pools of naturally occurring genetic elements can be cloned from genomicDNA or cDNA (Sambrook et al., 1989); for example, phage antibodylibraries, made by PCR amplification repertoires of antibody genes fromimmunised or unimrnunised donors have proved very effective sources offunctional antibody fragments (Winter et al., 1994; Hoogenboom, 1997).Libraries of genes can also be made by encoding all (see for exampleSmith, 1985; Parmley and Smith: 1988) or part of genes (see for exampleLowman et al., 1991) or pools of genes (see for example Nissim et al.,1994) by a randomised or doped synthetic oligonucleotide. Libraries canalso be made by introducing mutations into a genetic element or pool ofgenetic elements ‘randomly by a variety of techniques in vivo,including; using mutator strains of bacteria such as E. coli mutD5 (Liaoet al., 1986; Yamagishi et al., 1990; Low et at, 1996); using theantibody hypermutation system of B-lymphocytes (Yelamos et al., 1995).Random mutations can also be introduced both in vivo and in vitro bychemical mutagens, and ionising or UV irradiation (see Friedberg et al.,1995), or incorporation of mutagenic base analogues (Frees; 1959;Zaccolo et at, 1996). Random’ mutations can also be introduced intogenes in vitro during polymerisation for example by using error-pronepolymerases (Leung et al., 1989).

Further diversification can be introduced by using homologousrecombination either in vivo (see Kowalczykowski et al, 1994) or invitro (Stemmer, 1994a; Stemmer, 1994b).

According to a further aspect of the present invention, therefore, thereis provided a method of in vitro evolution comprising the steps of:

-   -   (a) selecting one or more genetic elements from a genetic        element library according to the present invention;    -   (b) mutating the selected genetic element(s) in order to        generate a further library of genetic elements encoding a        repertoire to gene products; and    -   (c) iteratively repeating steps (a) and (b) in order to obtain a        gene product with enhanced activity.

Mutations may be introduced into the genetic elements(s) as set forthabove.

The genetic elements according to the invention advantageously encodeenzymes, preferably of pharmacological or industrial interest,activators or inhibitors, especially of biological systems, such ascellular signal transduction mechanisms, antibodies and fragmentsthereof, and other binding agents (e.g. transcription factors) suitablefor diagnostic and therapeutic applications. In a preferred aspect,therefore, the invention permits the identification and isolation ofclinically or industrially useful products. In a further aspect of theinvention, there is provided a product when isolated by the method ofthe invention.

The selection of suitable encapsulation conditions is desirable.Depending on the complexity and size of the library to be screened, itmay be beneficial to set up the encapsulation procedure such that 1 orless than 1 genetic element is encapsulated per microcapsule. This willprovide the greatest power of resolution. Where the library is largerand/or more complex, however, this may be impracticable; it may bepreferable to encapsulate several genetic elements together and rely onrepeated application of the method of the invention to achieve sortingof the desired activity. A combination of encapsulation procedures maybe used to obtain the desired enrichment.

Theoretical studies indicate that the larger the number of geneticelement variants created the more likely it is that a molecule will becreated with the properties desired (see Perelson and Oster, 1979 for adescription of how this applies to repertoires of antibodies). Recentlyit has also been confirmed practically that larger phage-antibodyrepertoires do indeed give rise to more antibodies with better bindingaffinities than smaller repertoires (Griffiths et al., 1994). To ensurethat rare variants are generated and thus are capable of being selected,a large library size is desirable. Thus, the use of optimally smallmicrocapsules is beneficial.

The largest repertoire created to date using methods that require an invivo step (phage-display and Lad systems) has been a 1.6×10¹¹ clonephage-peptide library which required the fermentation of 15 litres ofbacteria (Fisch et al., 1996). SELEX experiments are often carried outon very large numbers of variants (up to 10¹⁵).

Using the present invention, at a preferred microcapsule diameter of 2.6μm, a repertoire size of at least 10¹¹ can be selected using 1 mlaqueous phase in a 20 ml emulsion.

In addition to the genetic elements described above, the microcapsulesaccording to the invention will comprise further components required forthe sorting process to take place. Other components of the system willfor example comprise those necessary for transcription and/ortranslation of the genetic element. These are selected for therequirements of a specific system from the following; a suitable buffer,an in vitro transcription/replication system and/or an in vitrotranslation system containing all the necessary ingredients, enzymes andcofactors, RNA polymerase, nucleotides, nucleic acids (natural orsynthetic), transfer RNAs, ribosomes and amino acids, and the substratesof the reaction of interest in order to allow selection of the modifiedgene product.

A suitable buffer will be one in which all of the desired components ofthe biological system are active and will therefore depend upon therequirements of each specific reaction system. Buffers suitable forbiological and/or chemical reactions are known in the art and recipesprovided in various laboratory texts, such as Sambrook et al., 1989.

The in vitro translation system will usually comprise a cell extract,typically from bacteria (Zubay, 1973; Zubay, 1980; Lesley et al., 1991;Lesley, 1995), rabbit reticulocytes (Pelham and Jackson, 1976), or wheatgerm (Anderson et al., 1983). Many suitable systems are commerciallyavailable (for example from Promega) including some which will allowcoupled transcription/translation (all the bacterial systems and thereticulocyte and wheat germ TNT™ extract systems from Promega). Themixture of amino acids used may include synthetic amino acids ifdesired, to increase the possible number or variety of proteins producedin the library. This can be accomplished by charging tRNAs withartificial amino acids and using these tRNAs for the in vitrotranslation of the proteins to be selected (Ellman et al., 1991; Benner,1994; Mendel et al., 1995).

After each round of selection the enrichment of the pool of geneticelements for those encoding the molecules of interest can be assayed bynon-compartmentalised in vitro transcription/replication or coupledtranscription-translation reactions. The selected pool is cloned into asuitable plasmid vector and RNA or recombinant protein is produced fromthe individual clones for further purification and assay.

In a preferred aspect, the internal environment of a microcapsule may bealtered by addition of reagents to the oil phase of the emulsion. Thereagents diffuse through the oil phase to the aqueous microcapsuleenvironment. Preferably, the reagents are at least partly water-soluble;such that a proportion thereof is distributed from the oil phase to theaqueous microcapsule environment. Advantageously, the reagents aresubstantially insoluble in the oil phase. Reagents are preferably mixedinto the oil phase by mechanical mixing, for example vortexing.

The reagents which may be added via the oil phase include substrates,buffering components, factors and the like. In particular, the internalpH of microcapsules may be altered in situ by adding acidic or basiccomponents to the oil phase.

The invention moreover relates to a method for producing a gene product,once a genetic element encoding the gene product has been sorted by themethod of the invention. Clearly, the genetic element itself may bedirectly expressed by conventional means to produce the gene product.However, alternative techniques may be employed, as will be apparent tothose skilled in the art. For example, the genetic informationincorporated in the gene product may be incorporated into a suitableexpression vector, and expressed therefrom.

The invention also describes the use of conventional screeningtechniques to identify compounds which are capable of interacting withthe gene products identified by the first aspect of the invention. Inpreferred embodiments, gene product encoding nucleic acid isincorporated into a vector, and introduced into suitable host cells toproduce transformed cell lines that express the gene product. Theresulting cell lines can then be produced for reproducible qualitativeand/or quantitative analysis of the effect(s) of potential drugsaffecting gene product function. Thus gene product expressing cells maybe employed for the identification of compounds, particularly smallmolecular weight compounds, which modulate the function of gene product.Thus host cells expressing gene product are useful for drug screeningand it is a further object of the present invention to provide a methodfor identifying compounds which modulate the activity of the geneproduct, said method comprising exposing cells containing heterologousDNA encoding gene product, wherein said cells produce functional geneproduct, to at least one compound or mixture of compounds or signalwhose ability to modulate the activity of said gene product is sought tobe determined, and thereafter monitoring said cells for changes causedby said modulation. Such an assay enables the identification ofmodulators, such as agonists, antagonists and allosteric modulators, ofthe gene product. As used herein, a compound or signal that modulatesthe activity of gene product refers to a compound that alters theactivity of gene product in such a way that the activity of the geneproduct is different in the presence of the compound or signal (ascompared to the absence of said compound or signal).

Cell-based screening assays can be designed by constructing cell linesin which the expression of a reporter protein, i.e. an easily assayableprotein, such as □-galactosidase, chloramphenicol acetyltransferase(CAT), green fluorescent protein (GFP) or luciferase, is dependent ongene product. Such an assay enables the detection of compounds thatdirectly modulate gene product function, such as compounds thatantagonise gene product, or compounds that inhibit or potentiate othercellular functions required for the activity of gene product.

The present invention also provides a method to exogenously affect geneproduct dependent processes occurring in cells. Recombinant gene productproducing host cells, e.g. mammalian cells, can be contacted with a testcompound, and the modulating effect(s) thereof can then be evaluated bycomparing the gene product-mediated response in the presence and absenceof test compound, or relating the gene product-mediated response of testcells, or control cells (i.e., cells that do not express gene product),to the presence of the compound.

In a further aspect, the invention relates to a method for optimising aproduction process which involves at least one step which is facilitatedby a polypeptide. For example, the step may be a catalytic step, whichis facilitated by an enzyme. Thus, the invention provides a method forpreparing a compound or compounds comprising the steps of:

-   -   (a) providing a synthesis protocol wherein at least one step is        facilitated by a polypeptide;    -   (b) preparing genetic elements encoding variants of the        polypeptide which facilitates this step, the expression of which        may result, directly or indirectly, in the modification of the        optical properties of the genetic elements;    -   (c) compartmentalising genetic elements into microcapsules;    -   (d) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (e) sorting the genetic elements which produce polypeptide gene        product(s) having the desired activity using the changed optical        properties of the genetic elements; and    -   (f) preparing the compound or compounds using the polypeptide        gene product identified in (g) to facilitate the relevant step        of the synthesis.

By means of the invention, enzymes involved in the preparation of acompound may be optimised by selection for optimal activity. Theprocedure involves the preparation of variants of the polypeptide to bescreened, which equate to a library of polypeptides as refereed toherein. The variants may be prepared in the same manner as the librariesdiscussed elsewhere herein.

The size of emulsion microcapsules may be varied simply by tailoring theemulsion conditions used to form the emulsion according to requirementsof the screening system. The larger the microcapsule size, the larger isthe volume that will .be required to encapsulate a given library, sincethe ultimately limiting factor will be the size of the microcapsule andthus the number of microcapsules possible per unit volume.

Water-in-oil emulsions can be re-emulsified to create water-in-oil-inwater double emulsions with an external (continuous) aqueous phase.These double emulsions can be analysed and, optionally, sorted using aflow cytometer (Bernath et al., 2004).

Highly monodisperse microcapsules can be produced using microfluidictechniques. For example, water-in-oil emulsions with less than 1.5%polydispersity can be generated by droplet break off in a co-flowingsteam of oil (Umbanhowar et al., 2000). Microfluidic systems can also beused for laminar-flow of aqueous microdroplets dispersed in a stream ofoil in microfluidic channels (Thorsen et al., 2001). This allows theconstruction of microfluidic devices for flow analysis and, optionally,flow sorting of microdroplets (Fu et al., 2002).

Advantageously, highly monodisperse microcapsules can be formed usingsystems and methods for the electronic control of fluidic species. Oneaspect of the invention relates to systems and methods for producingdroplets of fluid surrounded by a liquid. The fluid and the liquid maybe essentially immiscible in many cases, i.e., immiscible on a timescale of interest (e.g., the time it takes a fluidic droplet to betransported through a particular system or device). In certain cases,the droplets may each be substantially the same shape or size, asfurther described below. The fluid may also contain other species, forexample, certain molecular species (e.g., as further discussed below),cells, particles, etc.

In one set of embodiments, electric charge may be created on a fluidsurrounded by a liquid, which may cause the fluid to separate intoindividual droplets within the liquid. In some embodiments, the fluidand the liquid may be present in a channel, e.g., a microfluidicchannel, or other constricted space that facilitates application of anelectric field to the fluid (which may be “AC” or alternating current,“DC” or direct current etc.), for example, by limiting movement of thefluid with respect to the liquid. Thus, the fluid can be present as aseries of individual charged and/or electrically inducible dropletswithin the liquid. In one embodiment, the electric force exerted on thefluidic droplet may be large enough to cause the droplet to move withinthe liquid. In some cases, the electric force exerted on the fluidicdroplet may be used to direct a desired motion of the droplet within theliquid, for example, to or within a channel or a microfluidic channel(e.g., as further described herein), etc. As one example, in apparatus 5in FIG. 3A, droplets 15 created by fluid source 10 can be electricallycharged using an electric filed created by electric field generator 20.

Electric charge may be created in the fluid within the liquid using anysuitable technique, for example, by placing the fluid within an electricfield (which may be AC, DC, etc.), and/or causing a reaction to occurthat causes the fluid to have an electric charge, for example, achemical reaction, an ionic reaction, a photocatalyzed reaction, etc. Inone embodiment, the fluid is an electrical conductor. As used herein, a“conductor” is a material having a conductivity of at least about theconductivity of 18 megohm (MOhm or MΩ) water. The liquid surrounding thefluid may have a conductivity less than that of the fluid. For instance,the liquid may be an insulator, relative to the fluid, or at least a“leaky insulator,” i.e., the liquid is able to at least partiallyelectrically insulate the fluid for at least a short period of time.Those of ordinary skill in the art will be able to identify theconductivity of fluids. In one non-limiting embodiment, the fluid may besubstantially hydrophilic, and the liquid surrounding the fluid may besubstantially hydrophobic.

In some embodiments, the charge created on the fluid (for example, on aseries of fluidic droplets) may be at least about 10⁻²² C/micrometer³.In certain cases, the charge may be at least about 10⁻²¹ C/micrometer³,and in other cases, the charge may be at least about 10⁻²⁰C/micrometer³, at least about 10⁻¹⁹ C/micrometer³, at least about 10⁻¹⁸C/micrometer³, at least about 10⁻¹⁷ C/micrometer³, at least about 10⁻¹⁶C/micrometer³, at least about 10⁻¹⁵ C/micrometer³, at least about 10⁻¹⁴C/micrometer³, at least about 10⁻¹³ C/micrometer³, at least about 10⁻¹²C/micrometer³, at least about 10⁻¹¹ C/micrometer³, at least about 10⁻¹⁰C/micrometer³, or at least about 10⁻⁹ C/micrometer³ or more. In certainembodiments, the charge created on the fluid may be at least about 10⁻²¹C/micrometer², and in some cases, the charge may be at least about 10⁻²⁰C/micrometer², at least about 10⁻¹⁹ C/micrometer², at least about 10⁻¹⁸C/micrometer², at least about 10⁻¹⁷ C/micrometer², at least about 10⁻¹⁶C/micrometer², at least about 10⁻¹⁵ C/micrometer², at least about 10⁻¹⁴C/micrometer², or at least about 10⁻¹³ C/micrometer² or more. In otherembodiments, the charge may be at least about 10⁻¹⁴ C/droplet, and, insome cases, at least about 10⁻¹³ C/droplet, in other cases at leastabout 10⁻¹² C/droplet, in other cases at least about 10⁻¹¹ C/droplet, inother cases at least about 10⁻¹⁰ C/droplet, or in still other cases atleast about 10⁻⁹ C/droplet.

The electric field, in some embodiments, is generated from an electricfield generator, i.e., a device or system able to create an electricfield that can be applied to the fluid. The electric field generator mayproduce an AC field (i.e., one that varies periodically with respect totime, for example, sinusoidally, sawtooth, square, etc.), a DC field(i.e., one that is constant with respect to time), a pulsed field, etc.The electric field generator may be constructed and arranged to createan electric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments. As used herein, “integral” meansthat portions of the components integral to each other are joined insuch a way that the components cannot be manually separated from eachother without cutting or breaking at least one of the components.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof. In some cases, transparent or substantiallytransparent electrodes can be used. In certain embodiments, the electricfield generator can be constructed and arranged (e.g., positioned) tocreate an electric field applicable to the fluid of at least about 0.01V/micrometer, and, in some cases, at least about 0.03 V/micrometer, atleast about 0.05 V/micrometer, at least about 0.08 V/micrometer, atleast about 0.1 V/micrometer, at least about 0.3 V/micrometer, at leastabout 0.5 V/micrometer, at least about 0.7 V/micrometer, at least about1 V/micrometer, at least about 1.2 V/micrometer, at least about 1.4V/micrometer, at least about 1.6 V/micrometer, or at least about 2V/micrometer. In some embodiments, even higher electric fieldintensities may be used, for example, at least about 2 V/micrometer, atleast about 3 V/micrometer, at least about 5 V/micrometer, at leastabout 7 V/micrometer, or at least about 10 V/micrometer or more.

In some embodiments, an electric field may be applied to fluidicdroplets to cause the droplets to experience an electric force. Theelectric force exerted on the fluidic droplets may be, in some cases, atleast about 10⁻¹⁶ N/micrometer³. In certain cases, the electric forceexerted on the fluidic droplets may be greater, e.g., at least about10⁻¹⁵ N/micrometer³, at least about 10⁻¹⁴ N/micrometer³, at least about10⁻¹³ N/micrometer³, at least about 10⁻¹² N/micrometer³, at least about10⁻¹¹ N/micrometer³, at least about 10⁻¹⁰ N/micrometer³, at least about10⁻⁹ N/micrometer³, at least about 10⁻⁸ N/micrometer³, or at least about10⁻⁷ N/micrometer³ or more. In other embodiments, the electric forceexerted on the fluidic droplets, relative to the surface area of thefluid, may be at least about 10⁻¹⁵ N/micrometer², and in some cases, atleast about 10⁻¹⁴ N/micrometer², at least about 10⁻¹³ N/micrometer², atleast about 10⁻¹² N/micrometer², at least about 10⁻¹¹ N/micrometer², atleast about 10⁻¹⁰ N/micrometer², at least about 10⁻⁹ N/micrometer², atleast about 10⁻⁸ N/micrometer², at least about 10⁻⁷ N/micrometer², or atleast about 10⁻⁶ N/micrometer² or more. In yet other embodiments, theelectric force exerted on the fluidic droplets may be at least about10⁻⁹ N, at least about 10⁻⁸ N, at least about 10⁻⁷ N, at least about10⁻⁶ N, at least about 10⁻⁵ N, or at least about 10⁻⁴ N or more in somecases.

In some embodiments of the invention, systems and methods are providedfor at least partially neutralizing an electric charge present on afluidic droplet, for example, a fluidic droplet having an electriccharge, as described above. For example, to at least partiallyneutralize the electric charge, the fluidic droplet may be passedthrough an electric field and/or brought near an electrode, e.g., usingtechniques such as those described herein. Upon exiting of the fluidicdroplet from the electric field (i.e., such that the electric field nolonger has a strength able to substantially affect the fluidic droplet),and/or other elimination of the electric field, the fluidic droplet maybecome electrically neutralized, and/or have a reduced electric charge.

In another set of embodiments, droplets of fluid can be created from afluid surrounded by a liquid within a channel by altering the channeldimensions in a manner that is able to induce the fluid to formindividual droplets. The channel may, for example, be a channel thatexpands relative to the direction of flow, e.g., such that the fluiddoes not adhere to the channel walls and forms individual dropletsinstead, or a channel that narrows relative to the direction of flow,e.g., such that the fluid is forced to coalesce into individualdroplets. One example is shown in FIG. 7A, where channel 510 includes aflowing fluid 500 (flowing downwards), surrounded by liquid 505. Channel510 narrows at location 501, causing fluid 500 to form a series ofindividual fluidic droplets 515. In other embodiments, internalobstructions may also be used to cause droplet formation to occur. Forinstance, baffles, ridges, posts, or the like may be used to disruptliquid flow in a manner that causes the fluid to coalesce into fluidicdroplets.

In some cases, the channel dimensions may be altered with respect totime (for example, mechanically or electromechanically, pneumatically,etc.) in such a manner as to cause the formation of individual fluidicdroplets to occur. For example, the channel may be mechanicallycontracted (“squeezed”) to cause droplet formation, or a fluid streammay be mechanically disrupted to cause droplet formation, for example,through the use of moving baffles, rotating blades, or the like. As anon-limiting example, in FIG. 7B, fluid 500 flows through channel 510 ina downward direction. Fluid 500 is surrounded by liquid 505.Piezoelectric devices 520 positioned near or integral to channel 510 maythen mechanically constrict or “squeeze” channel 510, causing fluid 500to break up into individual fluidic droplets 515.

In yet another set of embodiments, individual fluidic droplets can becreated and maintained in a system comprising three essentially mutuallyimmiscible fluids (i.e., immiscible on a time scale of interest), whereone fluid is a liquid carrier, and the second fluid and the third fluidalternate as individual fluidic droplets within the liquid carrier. Insuch a system, surfactants are not necessarily required to ensureseparation of the fluidic droplets of the second and third fluids. As anexample, with reference to FIG. 14A, within channel 700, a first fluid701 and a second fluid 702 are each carried within liquid carrier 705.First fluid 701 and second fluid 702 alternate as a series ofalternating, individual droplets, each carried by liquid carrier 705within channel 700. As the first fluid, the second fluid, and the liquidcarrier are all essentially mutually immiscible, any two of the fluids(or all three fluids) can come into contact without causing dropletcoalescence to occur. A photomicrograph of an example of such a systemis shown in FIG. 14B, illustrating first fluid 701 and second fluid 702,present as individual, alternating droplets, each contained withinliquid carrier 705.

One example of a system involving three essentially mutually immisciblefluids is a silicone oil, a mineral oil, and an aqueous solution (i.e.,water, or water containing one or more other species that are dissolvedand/or suspended therein, for example, a salt solution, a salinesolution, a suspension of water containing particles or cells, or thelike). Another example of a system is a silicone oil, a fluorocarbonoil, and an aqueous solution. Yet another example of a system is ahydrocarbon oil (e.g., hexadecane), a fluorocarbon oil, and an aqueoussolution. In these examples, any of these fluids may be used as theliquid carrier. Non-limiting examples of suitable fluorocarbon oilsinclude octadecafluorodecahydro naphthalene:

or 1-(1,2,2,3,3,4,4,5,5,6,6-undecafluorocyclohexyl)ethanol:

A non-limiting example of such a system is illustrated in FIG. 14B. Inthis figure, fluidic network 710 includes a channel containing liquidcarrier 705, and first fluid 701 and second fluid 702. Liquid carrier705 is introduced into fluidic network 710 through inlet 725, whilefirst fluid 701 is introduced through inlet 721, and second fluid 702 isintroduced through inlet 722. Channel 716 within fluidic network 710contains liquid carrier 715 introduced from inlet 725. Initially, firstfluid 701 is introduced into liquid 10 carrier 705, forming fluidicdroplets therein. Next, second fluid 702 is introduced into liquid 705,forming fluidic droplets therein that are interspersed with the fluidicdroplets containing first fluid 701. Thus, upon reaching channel 717,liquid carrier 705 contains a first set of fluidic droplets containingfirst fluid 701, interspersed with a second set of fluidic dropletscontaining second fluid 702. In the embodiment illustrated, channel 706optionally comprises a series of bends, which may allow mixing to occurwithin each of the fluidic droplets, as further discussed below.However, it should be noted that in this embodiment, since first fluid701 and second fluid 702 are essentially immiscible, significant fusionand/or mixing of the droplets containing first fluid 701 with thedroplets containing second fluid 702 is not generally expected.

Other examples of the production of droplets of fluid surrounded by aliquid are described in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al. and InternationalPatent Application Serial No. PCT/US03/20542, filed Jun. 30, 2003 byStone, et al., published as WO 2004/002627 on Jan. 8, 2004, eachincorporated herein by reference.

In some embodiments, the fluidic droplets may each be substantially thesame shape and/or size. The shape and/or size can be determined, forexample, by measuring the average diameter or other characteristicdimension of the droplets. The term “determining,” as used herein,generally refers to the analysis or measurement of a species, forexample, quantitatively or qualitatively, and/or the detection of thepresence or absence of the species. “Determining” may also refer to theanalysis or measurement of an interaction between two or more species,for example, quantitatively or qualitatively, or by detecting thepresence or absence of the interaction. Examples of suitable techniquesinclude, but are not limited to, spectroscopy such as infrared,absorption, fluorescence, UV/visible, FTIR (“Fourier Transform InfraredSpectroscopy”), or Raman; gravimetric techniques; ellipsometry;piezoelectric measurements; immunoassays; electrochemical measurements;optical measurements such as optical density measurements; circulardichroism; light scattering measurements such as quasielectric lightscattering; polarimetry; refractometry; or turbidity measurements.

The “average diameter” of a plurality or series of droplets is thearithmetic average of the average diameters of each of the droplets.Those of ordinary skill in the art will be able to determine the averagediameter (or other characteristic dimension) of a plurality or series ofdroplets, for example, using laser light scattering, microscopicexamination, or other known techniques. The diameter of a droplet, in anon-spherical droplet, is the mathematically-defined average diameter ofthe droplet, integrated across the entire surface. The average diameterof a droplet (and/or of a plurality or series of droplets) may be, forexample, less than about 1 mm, less than about 500 micrometers, lessthan about 200 micrometers, less than about 100 micrometers, less thanabout 75 micrometers, less than about 50 micrometers, less than about 25micrometers, less than about 10 micrometers, or less than about 5micrometers in some cases. The average diameter may also be at leastabout 1 micrometer, at least about 2 micrometers, at least about 3micrometers, at least about 5 micrometers, at least about 10micrometers, at least about 15 micrometers, or at least about 20micrometers in certain cases.

In certain instances, the invention provides for the production ofdroplets consisting essentially of a substantially uniform number ofentities of a species therein (i.e., molecules, compounds, cells,genetic elements, particles, etc.). For example, about 90%, about 93%,about 95%, about 97%, about 98%, or about 99%, or more of a plurality orseries of droplets may each contain the same number of entities of aparticular species.

For instance, a substantial number of fluidic droplets produced, e.g.,as described above, may each contain 1 entity, 2 entities, 3 entities, 4entities, 5 entities, 7 entities, 10 entities, 15 entities, 20 entities,25 entities, 30 entities, 40 entities, 50 entities, 60 entities, 70entities, 80 entities, 90 entities, 100 entities, etc., where theentities are molecules or macromolecules, cells, particles, etc. In somecases, the droplets may each independently contain a range of entities,for example, less than 20 entities, less than 15 entities, less than 10entities, less than 7 entities, less than 5 entities, or less than 3entities in some cases. In one set of embodiments, in a liquidcontaining droplets of fluid, some of which contain a species ofinterest and some of which do not contain the species of interest, thedroplets of fluid may be screened or sorted for those droplets of fluidcontaining the species as further described below (e.g., usingfluorescence or other techniques such as those described above), and insome cases, the droplets may be screened or sorted for those droplets offluid containing a particular number or range of entities of the speciesof interest, e.g., as previously described. Thus, in some cases, aplurality or series of fluidic droplets, some of which contain thespecies and some of which do not, may be enriched (or depleted) in theratio of droplets that do contain the species, for example, by a factorof at least about 2, at least about 3, at least about 5, at least about10, at least about 15, at least about 20, at least about 50, at leastabout 100, at least about 125, at least about 150, at least about 200,at least about 250, at least about 500, at least about 750, at leastabout 1000, at least about 2000, or at least about 5000 or more in somecases. In other cases, the enrichment (or depletion) may be in a ratioof at least about 10⁴, at least about 10⁵, at least about 10⁶, at leastabout 10⁷, at least about 10⁸, at least about 10⁹, at least about 10¹⁰,at least about 10¹¹, at least about 10¹², at least about 10¹³, at leastabout 10¹⁴, at least about 10¹⁵, or more. For example, a fluidic dropletcontaining a particular species may be selected from a library offluidic droplets containing various species, where the library may haveabout 10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹, about 10¹⁰, about10¹¹, about 10¹², about 10¹³, about 10¹⁴, about 10¹⁵, or more items, forexample, a DNA library, an RNA library, a protein library, acombinatorial chemistry library, a library of genetic elements, etc. Incertain embodiments, the droplets carrying the species may then befused, reacted, or otherwise used or processed, etc., as furtherdescribed below, for example, to initiate or determine a reaction.

The use of microfluidic handling to create microcapsoules according tothe invention has a number of advantages:

-   -   (a) They allow the formation of highly monodisperse        microcapsules (<1.5% polydispersity), each of which functions as        an almost identical, very small microreactor;    -   (b) The microcapsules can have volumes ranging from about 1        femtolitre to about 1 nanolitre;    -   (c) Compartmentalisation in microcapsules prevents diffusion and        dispersion due to parabolic flow;    -   (d) By using a perfluorocarbon carrier fluid it is possible to        prevent exchange of molecules between microcapsules;    -   (e) Reagents in microcapsules cannot react or interact with the        fabric of the microchannels as they are separated by a layer of        inert perfluorocarbon carrier fluid.    -   (f) Microcapsules can be created at up to 10,000 per second and        screened using optical methods at the same rate. This is a        throughput of 10⁹ per day.

Microcapsules (or droplets; the terms may be used intechangeably for thepurposes envisaged herein) can, advantageously, be fused or split. Forexample, aqueous microdroplets can be merged and split usingmicrofluidics systems (Link et al., 2004; Song et al., 2003).Microcapsule fusion allows the mixing of reagents. Fusion, for example,of a microcapsule containing the genetic element with a microcapsulecontaining a transcription factor could initiate transcription of thegenetic information. Microcapsule splitting allows single microcapsulesto be split into two or more smaller microcapsules. For example a singlemicrocapsule containing a ragent can be split into multiplemicrocapsules which can then each be fused with a different microcapsulecontaining a different reagent or genetic element. A single microcapsulecontaining a reagent can also be split into multiple microcapsules whichcan then each be fused with a different microcapsule containing adifferent genetic element, or other reagents, for example at differentconcentrations.

In one aspect, the invention relates to microfluidic systems and methodsfor splitting a fluidic droplet into two or more droplets. The fluidicdroplet may be surrounded by a liquid, e.g., as previously described,and the fluid and the liquid are essentially immiscible in some cases.The two or more droplets created by splitting the original fluidicdroplet may each be substantially the same shape and/or size, or the twoor more droplets may have different shapes and/or sizes, depending onthe conditions used to split the original fluidic droplet. In manycases, the conditions used to split the original fluidic droplet can becontrolled in some fashion, for example, manually or automatically(e.g., with a processor, as discussed below). In some cases, eachdroplet in a plurality or series of fluidic droplets may beindependently controlled. For example, some droplets may be split intoequal parts or unequal parts, while other droplets are not split.

According to one set of embodiments, a fluidic droplet can be splitusing an applied electric field. The electric field may be an AC field,a DC field, etc. The fluidic droplet, in this embodiment, may have agreater electrical conductivity than the surrounding liquid, and, insome cases, the fluidic droplet may be neutrally charged. In someembodiments, the droplets produced from the original fluidic droplet areof approximately equal shape and/or size. In certain embodiments, in anapplied electric field, electric charge may be urged to migrate from theinterior of the fluidic droplet to the surface to be distributedthereon, which may thereby cancel the electric field experienced in theinterior of the droplet. In some embodiments, the electric charge on thesurface of the fluidic droplet may also experience a force due to theapplied electric field, which causes charges having opposite polaritiesto migrate in opposite directions. The charge migration may, in somecases, cause the drop to be pulled apart into two separate fluidicdroplets. The electric field applied to the fluidic droplets may becreated, for example, using the techniques described above, such as witha reaction an electric field generator, etc.

As a non-limiting example, in FIG. 1A, where no electric field isapplied, fluidic droplets 215 contained in channel 230 are carried by asurrounding liquid, which.flows towards intersection 240, leading tochannels 250 and 255. In this example, the surrounding liquid flowsthrough channels 250 and 255 at equal flowrates. Thus, at intersection240, fluidic droplets 215 do not have a preferred orientation ordirection, and move into exit channels 250 and 255 with equalprobability due to the surrounding liquid flow. In contrast, in FIG. 1B,while the surrounding liquid flows in the same fashion as FIG. 1A, underthe influence of an applied electric field of 1.4 V/micrometers, fluidicdroplets 215 are split into two droplets at intersection 240, formingnew droplets 216 and 217. Droplet 216 moves to the left in channel 250,while droplet 217 moves to the right in channel 255.

A schematic of this process can be seen in FIG. 5, where a neutralfluidic droplet 530, surrounded by a liquid 535 in channel 540, issubjected to applied electric field 525, created by electrodes 526 and527. Electrode 526 is positioned near channel 542, while electrode 527is positioned near channel 544. Under the influence of electric field525, charge separation is induced within fluidic droplet 530, i.e., suchthat a positive charge is induced at one end of the droplet, while anegative charge is induced at the other end of the droplet. The dropletmay then split into a negatively charged droplet 545 and a positivelycharged droplet 546, which then may travel in channels 542 and 544,respectively. In some cases, one or both of the electric charges on theresulting charged droplets may also be neutralized, as previouslydescribed.

Other examples of splitting a fluidic droplet into two droplets aredescribed in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al.; U.S. ProvisionalPatent Application Ser. No. 60/498,091, filed Aug. 27, 2003, by Link,et. al.; and International Patent Application Serial No. PCT/US03/20542,filed Jun. 30, 2003 by Stone, et al., published as WO 2004/002627 onJan. 8, 2004, each incorporated herein by reference.

The invention, in yet another aspect, relates to systems and methods forfusing or coalescing two or more fluidic droplets into one droplet. Forexample, in one set of embodiments, systems and methods are providedthat are able to cause two or more droplets (e.g., arising fromdiscontinuous streams of fluid) to fuse or coalesce into one droplet incases where the two or more droplets ordinarily are unable to fuse orcoalesce, for example, due to composition, surface tension, dropletsize, the presence or absence of surfactants, etc. In certainmicrofluidic systems, the surface tension of the droplets, relative tothe size of the droplets, may also prevent fusion or coalescence of thedroplets from occurring in some cases.

In one embodiment, two fluidic droplets may be given opposite electriccharges (i.e., positive and negative charges, not necessarily of thesame magnitude), which may increase the electrical interaction of thetwo droplets such that fusion or coalescence of the droplets can occurdue to their opposite electric charges, e.g., using the techniquesdescribed herein. For instance, an electric field may be applied to thedroplets, the droplets may be passed through a capacitor, a chemicalreaction may cause the droplets to become charged, etc. As an example,as is shown schematically in FIG. 13A, uncharged droplets 651 and 652,carried by a liquid 654 contained within a microfluidic channel 653, arebrought into contact with each other, but the droplets are not able tofuse or coalesce, for instance, due to their size and/or surfacetension. The droplets, in some cases, may not be able to fuse even if asurfactant is applied to lower the surface tension of the droplets.However, if the fluidic droplets are electrically charged with oppositecharges (which can be, but are not necessarily of, the same magnitude),the droplets may be able to fuse or coalesce. For instance, in FIG. 13B,positively charged droplets 655 and negatively charged droplets 656 aredirected generally towards each other such that the electricalinteraction of the oppositely charged droplets causes the droplets tofuse into fused droplets 657.

In another embodiment, the fluidic droplets may not necessarily be givenopposite electric charges (and, in some cases, may not be given anyelectric charge), and are fused through the use of dipoles induced inthe fluidic droplets that causes the fluidic droplets to coalesce. Inthe example illustrated in FIG. 13C, droplets 660 and 661 (which mayeach independently be electrically charged or neutral), surrounded byliquid 665 in channel 670, move through the channel such that they arethe affected by an applied electric field 675. Electric field 675 may bean AC field, a DC field, etc., and may be created, for instance, usingelectrodes 676 and 677, as shown here. The induced dipoles in each ofthe fluidic droplets, as shown in FIG. 13C, may cause the fluidicdroplets to become electrically attracted towards each other due totheir local opposite charges, thus causing droplets 660 and 661 to fuseto produce droplet 663. In FIG. 13D, droplets 651 and 652 flow togetherto fuse to form droplet 653, which flows in a third channel.

It should be noted that, in various embodiments, the two or moredroplets allowed to coalesce are not necessarily required to meet“head-on”. Any angle of contact, so long as at least some fusion of thedroplets initially occurs, is sufficient. As an example, in FIG. 12H,droplets 73 and 74 each are traveling in substantially the samedirection (e.g., at different velocities), and are able to meet andfuse. As another example, in FIG. 12I, droplets 73 and 74 meet at anangle and fuse. In FIG. 12J, three fluidic droplets 73, 74 and 68 meetand fuse to produce droplet 79.

Other examples of fusing or coalescing fluidic droplets are described inInternational Patent Application Serial No. PCT/US2004/010903, filedApr. 9, 2004 by Link, et al., incorporated herein by reference.

Fluidic handling of microcapsules therefore results in furtheradvantages:

-   -   (a) Microcapsules can be split into two or more smaller        microdroplets allowing the reagents contained therein to be        reacted with a series of different molecules in parallel or        assayed in multiplicate.    -   (b) Microcapsules can be fused. This allows molecules to be: (a)        diluted, (b) mixed with other molecules, and (c) reactions        initiated, terminated or modulated at precisely defined times.    -   (c) Reagents can be mixed very rapidly (in <2 ms) in        microcapsules using chaotic advection, allowing fast kinetic        measurements and very high throughput.    -   (d) Reagents can be mixed in a combinatorial manner. For        example, allowing the effect of all possible pairwise        combinations of compounds in a library to be tested.

Creating and manipulating microcapsules in microfluidic systems meansthat:

-   -   (a) Stable streams of microcapsules can be formed in        microchannels and identified by their relative positions.    -   (b) If the reactions are accompanied by an optical signal (e.g.        a change in fluorescence) a spatially-resolved optical image of        the microfluidic network allows time resolved measurements of        the reactions in each microcapsules.    -   (c) Microcapsules can be separated using a microfluidic flow        sorter to allow recovery and further analysis or manipulation of        the molecules they contain.

Screening/Sorting of Microcapsules

In still another aspect, the invention provides systems and methods forscreening or sorting fluidic droplets in a liquid, and in some cases, atrelatively high rates. For example, a characteristic of a droplet may besensed and/or determined in some fashion (e.g., as further describedbelow), then the droplet may be directed towards a particular region ofthe device, for example, for sorting or screening purposes.

In some embodiments, a characteristic of a fluidic droplet may be sensedand/or determined in some fashion, for example, as described herein(e.g., fluorescence of the fluidic droplet may be determined), and, inresponse, an electric field may be applied or removed from the fluidicdroplet to direct the fluidic droplet to a particular rgion (e.g. achannel). In some cases, high sorting speeds may be achievable usingcertain systems and methods of the invention. For instance, at leastabout 10 droplets per second may be determined and/or sorted in somecases, and in other cases, at least about 20 droplets per second, atleast about 30 droplets per second, at least about 100 droplets persecond, at least about 200 droplets per second, at least about 300droplets per second, at least about 500 droplets per second, at leastabout 750 droplets per second, at least about 1000 droplets per second,at least about 1500 droplets per second, at least about 2000 dropletsper second, at least about 3000 droplets per second, at least about 5000droplets per second, at least about 7500 droplets per second, at leastabout 10,000 droplets per second, at least about 15,000 droplets persecond, at least about 20,000 droplets per second, at least about 30,000droplets per second, at least about 50,000 droplets per second, at leastabout 75,000 droplets per second, at least about 100,000 droplets persecond, at least about 150,000 droplets per second, at least about200,000 droplets per second, at least about 300,000 droplets per second,at least about 500,000 droplets per second, at least about 750,000droplets per second, at least about 1,000,000 droplets per second, atleast about 1,500,000 droplets per second, at least about 2,000,000 ormore droplets per second, or at least about 3,000,000 or more dropletsper second may be determined and/or sorted in such a fashion.

In one set of embodiments, a fluidic droplet may be directed by creatingan electric charge (e.g., as previously described) on the droplet, andsteering the droplet using an applied electric field, which may be an ACfield, a DC field, etc. As an example, in reference to FIGS. 2-4, anelectric field may be selectively applied and removed (or a differentelectric field may be applied, e.g., a reversed electric field as shownin FIG. 4A) as needed to direct the fluidic droplet to a particularregion. The electric field may be selectively applied and removed asneeded, in some embodiments, without substantially altering the flow ofthe liquid containing the fluidic droplet. For example, a liquid mayflow on a substantially steady-state basis (i.e., the average flowrateof the liquid containing the fluidic droplet deviates by less than 20%or less than 15% of the steady-state flow or the expected value of theflow of liquid with respect to time, and in some cases, the averageflowrate may deviate less than 10% or less than 5%) or otherpredetermined basis through a fluidic system of the invention (e.g.,through a channel or a microchannel), and fluidic droplets containedwithin the liquid may be directed to various regions, e.g., using anelectric field, without substantially altering the flow of the liquidthrough the fluidic system. As a particular example, in FIGS. 2A, 3A and4A, a liquid containing fluidic droplets 15 flows from fluid source 10,through channel 30 to intersection 40, and exits through channels 50 and55. In FIG. 2A, fluidic droplets 15 are directed through both channels50 and 55, while in FIG. 3A, fluidic droplets 15 are directed to onlychannel 55 and, in FIG. 4A, fluidic droplets 15 are directed to onlychannel 50.

In another set of embodiments, a fluidic droplet may be sorted orsteered by inducing a dipole in the fluidic droplet (which may beinitially charged or uncharged), and sorting or steering the dropletusing an applied electric field. The electric field may be an AC field,a DC field, etc. For example, with reference to FIG. 9A, a channel 540,containing fluidic droplet 530 and liquid 535, divides into channel 542and 544. Fluidic droplet 530 may have an electric charge, or it may beuncharged. Electrode 526 is positioned near channel 542, while electrode527 is positioned near channel 544. Electrode 528 is positioned near thejunction of channels 540, 542, and 544. In FIGS. 9C and 9D, a dipole isinduced in the fluidic droplet using electrodes 526, 527, and/or 528. InFIG. 9C, a dipole is induced in droplet 530 by applying an electricfield 525 to the droplet using electrodes 527 and 528. Due to thestrength of the electric field, the droplet is strongly attracted to theright, into channel 544. Similarly, in FIG. 9D, a dipole is induced indroplet 530 by applying an electric field 525 to the droplet usingelectrodes 526 and 528, causing the droplet to be attracted into channel542. Thus, by applying the proper electric field, droplet 530 can bedirected to either channel 542 or 544 as desired.

In other embodiments, however, the fluidic droplets may be screened orsorted within a fluidic system of the invention by altering the flow ofthe liquid containing the droplets. For instance, in one set ofembodiments, a fluidic droplet may be steered or sorted by directing theliquid surrounding the fluidic droplet into a first channel, a secondchannel, etc. As a non-limiting example, with reference to FIG. 10A,fluidic droplet 570 is surrounded by a liquid 575 in channel 580.Channel 580 divides into three channels 581, 582, and 583. The flow ofliquid 575 can be directed into any of channels 581, 582, and 583 asdesired, for example, using flow-controlling devices known to those ofordinary skill in the art, for example, valves, pumps, pistons, etc.Thus, in FIG. 10B, fluidic droplet 570 is directed into channel 581 bydirecting liquid 575 to flow into channel 581 (indicated by arrows 574);in FIG. 10C, fluidic droplet 570 is directed into channel 582 bydirecting liquid 575 to flow into channel 582 (indicated by arrows 574);and in FIG. 10D, fluidic droplet 570 is directed into channel 583 bydirecting liquid 575 to flow into channel 583 (indicated by arrows 574).

However, it is preferred that control of the flow of liquids inmicrofluidic systems is not used to direct the flow of fluidic dropletstherein, but that an alternative method is used. Advantageously,therefore, the microcapsules are not sorted by altering the direction ofthe flow of a carrier fluid in a microfluidic system.

In another set of embodiments, pressure within a fluidic system, forexample, within different channels or within different portions of achannel, can be controlled to direct the flow of fluidic droplets. Forexample, a droplet can be directed toward a channel junction includingmultiple options for further direction of flow (e.g., directed toward abranch, or fork, in a channel defining optional downstream flowchannels). Pressure within one or more of the optional downstream flowchannels can be controlled to direct the droplet selectively into one ofthe channels, and changes in pressure can be effected on the order ofthe time required for successive droplets to reach the junction, suchthat the downstream flow path of each successive droplet can beindependently controlled. In one arrangement, the expansion and/orcontraction of liquid reservoirs may be used to steer or sort a fluidicdroplet into a channel, e.g., by causing directed movement of the liquidcontaining the fluidic droplet. The liquid reservoirs may be positionedsuch that, when activated, the movement of liquid caused by theactivated reservoirs causes the liquid to flow in a preferred direction,carrying the fluidic droplet in that preferred direction. For instance,the expansion of a liquid reservoir may cause a flow of liquid towardsthe reservoir, while the contraction of a liquid reservoir may cause aflow of liquid away from the reservoir. In some cases, the expansionand/or contraction of the liquid reservoir may be combined with otherflow-controlling devices and methods, e.g., as described herein.Non-limiting examples of devices able to cause the expansion and/orcontraction of a liquid reservoir include pistons and piezoelectriccomponents. In some cases, piezoelectric components may be particularlyuseful due to their relatively rapid response times, e.g., in responseto an electrical signal.

As a non-limiting example, in FIG. 11A, fluidic droplet 600 issurrounded by a liquid 605 in channel 610. Channel 610 divides intochannels 611, 612. Positioned in fluidic communication with channels 611and 612 are liquid reservoirs 617 and 618, which may be expanded and/orcontracted, for instance, by piezoelectric components 615 and 616, by apiston (not shown), etc. In FIG. 11B, liquid reservoir 617 has beenexpanded, while liquid reservoir 618 has been contracted. The effect ofthe expansion/contractions of the reservoirs is to cause a net flow ofliquid towards channel 611, as indicated by arrows 603. Thus, fluidicdroplet 600, upon reaching the junction between the channels, isdirected to channel 611 by the movement of liquid 605. The reversesituation is shown in FIG. 11C, where liquid reservoir 617 hascontracted while liquid reservoir 618 has been expanded. A net flow ofliquid occurs towards channel 612 (indicated by arrows 603), causingfluidic droplet 600 to move into channel 612. It should be noted,however, that reservoirs 617 and 618 do not both need to be activated todirect fluidic droplet 600 into channels 611 or 612. For example, in oneembodiment, fluidic droplet 600 may be directed to channel 611 by theexpansion of liquid reservoir 617 (without any alteration of reservoir618), while in another embodiment, fluidic droplet 600 may be directedto channel 611 by the contraction of liquid reservoir 618 (without anyalteration of reservoir 617). In some cases, more than two liquidreservoirs may be used.

In some embodiments, the fluidic droplets may be sorted into more thantwo channels. Non-limiting examples of embodiments of the inventionhaving multiple regions within a fluidic system for the delivery ofdroplets are shown in FIGS. 6A and 6B. Other arrangements are shown inFIGS. 10A-10D. In FIG. 6A, charged droplets 315 in channel 330 may bedirected as desired to any one of exit channels 350, 352, 354, or 356,by applying electric fields to control the movement of the droplets atintersections 340, 341, and 342, using electrodes 321/322, 323/324, and325/326, respectively. In FIG. 6A, droplets 315 are directed to channel354 using applied electric fields 300 and 301, using 5 principlessimilar to those discussed above. Similarly, in FIG. 6B, chargeddroplets 415 in channel 430 can be directed to any one of exit channels450, 452, 454, 456, or 458, by applying electric fields to control themovement of the droplets at intersections 440, 441, 442, and 443, usingelectrodes 421/422, 423/424, 425/426, and 427/428, respectively. In thisfigure, droplets 415 are directed to channel 454; of course, the chargeddroplets may be directed to any other exit channel as desired.

In another example, in apparatus 5, as schematically illustrated in FIG.2A, fluidic droplets 15 created by fluid source 10 are positivelycharged due to an applied electric field created using electric fieldgenerator 20, which comprises two electrodes 22, 24. Fluidic droplets 15are directed through channel 30 by a liquid containing the droplets, andare directed towards intersection 40. At intersection 40, the fluidicdroplets do not have a preferred orientation or direction, and move intoexit channels 50 and 55 with equal probability (in this embodiment,liquid drains through both exit channels 50 and 55 at substantiallyequal rates). Similarly, fluidic droplets 115 created by fluid source110 are negatively charged due to an applied electric field createdusing electric field generator 120, which comprises electrodes 122 and124. After traveling through channel 130 towards intersection 140, thefluidic droplets do not have a preferred orientation or direction, andmove into exit channels 150 and 155 with equal probability, as theliquid exits through exit channels 150 and 155 at substantially equalrates. A representative photomicrograph of intersection 140 is shown inFIG. 2B.

In the schematic diagram of FIG. 3A, an electric field 100 of 1.4V/micrometer has been applied to apparatus 5 of FIG. 2A, in a directiontowards the right of apparatus 5. Positively-charged fluidic droplets 15in channel 30, upon reaching intersection 40, are directed to the rightin channel 55 due to the applied electric field 100, while the liquidcontaining the droplets continues to exit through exit channels 50 and55 at substantially equal rates. Similarly, negatively-charged fluidicdroplets 115 in channel 130, upon reaching intersection 140, aredirected to the left in channel 150 due to the applied electric field100, while the liquid fluid continues to exit the device through exitchannels 150 and 155 at substantially equal rates. Thus, electric field100 can be used to direct fluidic droplets into particular channels asdesired. A representative photomicrograph of intersection 140 is shownin FIG. 3B.

FIG. 4A is a schematic diagram of apparatus 5 of FIG. 2A, also with anapplied electric field 100 of 1.4 V/micrometer, but in the oppositedirection (i.e., −1.4 V/micrometer). In this figure, positively-chargedfluidic droplets 15 in channel 30, upon reaching intersection 40, aredirected to the left into channel 50 due to the applied electric field100, while negatively-charged fluidic droplets 115 in channel 130, uponreaching intersection 140, are directed to the right into channel 155due to applied electric field 100. The liquid containing the dropletsexits through exit channels 50 and 55, and 150 and 155, at substantiallyequal rates. A representative photomicrograph of intersection 140 isshown in FIG. 4B.

In some embodiments of the invention, a fluidic droplet may be sortedand/or split into two or more separate droplets, for example, dependingon the particular application. Any of the above-described techniques maybe used to spilt and/or sort droplets. As a non-limiting example, byapplying (or removing) a first electric field to a device (or a portionthereof), a fluidic droplet may be directed to a first region orchannel; by applying (or removing) a second electric field to the device(or a portion thereof), the droplet may be directed to a second regionor channel; by applying a third electric field to the device (or aportion thereof), the droplet may be directed to a third region orchannel; etc., where the electric fields may differ in some way, forexample, in intensity, direction, frequency, duration, etc. In a seriesof droplets, each droplet may be independently sorted and/or split; forexample, some droplets may be directed to one location or another, whileother droplets may be split into multiple droplets directed to two ormore locations.

As one particular example, in FIG. 8A, fluidic droplet 550, surroundingliquid 555 in channel 560 may be directed to channel 556, channel 557,or be split in some fashion between channels 562 and 564. In FIG. 8B, bydirecting surrounding liquid 555 towards channel 562, fluidic droplet550 may be directed towards the left into channel 562; in FIG. 8C, bydirecting surrounding liquid 555 towards channel 564, fluidic droplet550 may be directed towards the right into channel 564, In FIG. 8D, anelectric field may be applied, in combination with control of the flowof liquid 555 surrounding fluidic droplet 550, that causes the dropletto impact junction 561, which may cause the droplet to split into twoseparate fluidic droplets 565, 566. Fluidic droplet 565 is directed tochannel 562, while fluidic droplet 566 is directed to channel 566. Ahigh degree of control of the applied electric field may be achieved tocontrol droplet formation; thus, for example, after fluidic droplet 565has been split into droplets 565 and 566, droplets 565 and 566 may be ofsubstantially equal size, or either of droplets 565 and 566 may belarger, e.g., as is shown in FIGS. 8E and 8F, respectively.

As another, example, in FIG. 9A, channel 540, carrying fluidic droplet530 and liquid 535, divides into channel 542 and 544. Fluidic droplet530 may be electrically charged, or it may uncharged. Electrode 526 ispositioned near channel 542, while electrode 527 is positioned nearchannel 544. Electrode 528 is positioned near the junction of channels540, 542, and 544. When fluidic droplet 530 reaches the junction, it maybe subjected to an electric field, and/or directed to a channel or otherregion, for example, by directing the surrounding liquid into thechannel. As shown in FIG. 9B, fluidic droplet 530 may be split into twoseparate droplets 565 and 566 by applying an electric field 525 to thedroplet using electrodes 526 and 527. In FIG. 9C, a dipole can beinduced in droplet 530 by applying an electric field 525 to the dropletusing electrodes 527 and 528. Due to the strength of the appliedelectric field, the droplet may be strongly attracted to the right, intochannel 544. Similarly, in FIG. 9D, a dipole may be induced in droplet530 by applying an electric field 525 to the droplet using electrodes526 and 528, causing the droplet to be attracted into channel 542. Bycontrolling which electrodes are used to induce an electric field acrossdroplet 530, and/or the strength of the applied electric field, one ormore fluidic droplets within channel 540 may be sorted and/or split intotwo droplets, and each droplet may independently be sorted and/or split.

Microcapsules can be optically tagged by, for example, incorporatingfluorochromes. In a preferred configuration, the microcapsules areoptically tagged by incorporating quantum dots: quantum dots of 6colours at 10 concentrations would allow the encoding of 10⁶microcapsules (Han et al., 2001). Microcapsules flowing in an orderedsequence in a microfluidic channel can be encoded (wholly or partially)by their sequence in the stream of microcapsules (positional encoding).

By means of the invention, enzymes involved in the preparation of acompound may be optimised by selection for optimal activity. Theprocedure involves the preparation of variants of the polypeptide to bescreened, which equate to a library of polypeptides as refereed toherein. The variants may be prepared in the same manner as the librariesdiscussed elsewhere herein.

(B) Selection Procedures

The system can be configured to select for RNA, DNA or protein geneproduct molecules with catalytic, regulatory or binding activity.

(i) Selection for Binding

In the case of selection for a gene product with affinity for a specificligand the genetic element may be linked to the gene product in themicrocapsule via the ligand. Only gene products with affinity for theligand will therefore bind to the genetic element and only those geneticelements with gene product bound via the ligand will acquire the changedoptical properties which enable them to be retained in the selectionstep. In this embodiment, the genetic element will thus comprise anucleic acid encoding the gene product linked to a ligand for the geneproduct.

The change in optical properties of the genetic element after binding ofthe gene product to the ligand may be induced in a variety of ways,including:

-   -   (1) the gene product itself may have distinctive optical        properties, for example, it is fluorescent (e.g. green        fluorescent protein, (Lorenz et al., 1991)).    -   (2) the optical properties of the gene product may be modified        on binding to the ligand, for example, the fluorescence of the        gene product is quenched or enhanced on binding (Guixe et al.,        1998; Qi and Grabowski, 1998)    -   (3) the optical properties of the ligand may be modified on        binding of the gene product, for example, the fluorescence of        the ligand is quenched or enhanced on binding (Voss, 1993; Masui        and Kuramitsu, 1998).    -   (4) the optical properties of both ligand and gene product are        modified on binding, for example, there can be a fluorescence        resonance energy transfer (FRET) from ligand to gene product (or        vice versa) resulting in emmission at the “acceptor” emmission        wavelength when excitation is at the “donor” absoption        wavelength (Heim & Tsien, 1996; Mahaj an et al., 1998; Miyawaki        et al., 1997).

In this embodiment, it is not necessary for binding of the gene productto the genetic element via the ligand to directly induce a change inoptical properties. All the gene products to be selected can contain aputative binding domain, which is to be selected for, and a commonfeature—a tag. The genetic element in each microcapsule is physicallylinked to the ligand. If the gene product produced from the geneticelement has affinity for the ligand, it will bind to it and becomephysically linked to the same genetic element that encoded it, resultingin the genetic element being ‘tagged’. At the end of the reaction, allof the microcapsules are combined, and all genetic elements and geneproducts pooled together in one environment. Genetic elements encodinggene products exhibiting the desired binding can be selected by addingreagents which specifically bind to, or react specifically with, the“tag” and thereby induce a change in the optical properties of thegenetic element allowing there sorting. For example, afluorescently-labelled anti-“tag” antibody can be used, or an anti-“tag”antibody followed by a second fluorescently labelled antibody whichbinds the first.

In an alternative embodiment, genetic elements may be sorted on thebasis that the gene product, which binds to the ligand, merely hides theligand from, for example, further binding partners which would otherwisemodify the optical properties of the genetic element. In this casegenetic elements with ummodified optical properties would be selected.

In an alternative embodiment, the invention provides a method accordingto the first aspect of the invention, wherein in step (b) the geneproducts bind to genetic elements encoding them. The gene productstogether with the attached genetic elements are then sorted as a resultof binding of a ligand to gene products having the desired bindingactivity. For example, all gene products can contain an invariant regionwhich binds covalently or non-covalently to the genetic element, and asecond region which is diversified so as to generate the desired bindingactivity.

In an alternative embodiment, the ligand for the gene product is itselfencoded by the genetic element and binds to the genetic element. Statedotherwise, the genetic element encodes two (or indeed more) geneproducts, at least one of which binds to the genetic element, and whichcan potentially bind each other. Only when the gene products interact ina microcapsule is the genetic element modified in a way that ultimatelyresults in a change in a change in its optical properties that enablesit to be sorted. This embodiment, for example, isused to search genelibraries for pairs of genes encoding pairs of proteins which bind eachother.

Fluorescence may be enhanced by the use of Tyramide Signal Amplification(TSA™) amplification to make the genetic elements fluorescent. Thisinvolves peroxidase (linked to another protein) binding to the geneticelements and catalysing the conversion of fluorescein-tyramine in to afree radical form which then reacts (locally) with the genetic elements.Methods for performing TSA are known in the art, and kits are availablecommercially from NEN.

TSA may be configured such that it results in a direct increase in thefluorescence of the genetic element, or such that a ligand is attachedto the genetic element which is bound by a second fluorescent molecule,or a sequence of molecules, one or raore of which is fluorescent.

(ii) Selection for Catalysis

When selection is for catalysis, the genetic element in eachmicrocapsule may comprise the substrate of the reaction. If the geneticelement encodes a gene product capable of acting as a catalyst, the geneproduct will catalyse the conversion of the substrate into the product.Therefore, at the end of the reaction the genetic element is physicallylinked to the product of the catalysed reaction.

It may also be desirable, in some cases, for the substrate not to be acomponent of the genetic element. In this case the substrate wouldcontain an inactive “tag” that requires a further step to activate itsuch as photoactivation (e.g. of a “caged” biotin analogue, (Sundberg etal., 1995; Pirrung and Huang, 1996)). The catalyst to be selected thenconverts the substrate to product. The “tag” is then activated and the“tagged” substrate and/or product bound by a tag-binding molecule (e.g.avidin or streptavidin) complexed with the nucleic acid. The ratio ofsubstrate to product attached to the nucleic acid via the “tag” willtherefore reflect the ratio of the substrate and product in solution.

The optical properties of genetic elements with product attached andwhich encode gene products with the desired catalytic activity can bemodified by either:

-   -   (1) the product-genetic element complex having characteristic        optical properties not found in the substrate-genetic element        complex, due to, for example;        -   (a) the substrate and product having different optical            properties (many fluorogenic enzyme substrates are available            commercially (see for example Haugland, 1996) including            substrates for glycosidases, phosphatases, peptidases and            proteases (Craig et al., 1995; Huang et al., 1992; Brynes et            al., 1982; Jones et al., 1997; Matayoshi et al., 1990; Wang            et al., 1990)), or        -   (b) the substrate and product having similar optical            properties, but only the product, and not the substrate            binds to, or reacts with, the genetic element;    -   (2) adding reagents which specifically bind to, or react with,        the product and which thereby induce a change in the optical        properties of the genetic elements allowing their sorting (these        reagents can be added before or after breaking the microcapsules        and pooling the genetic elements). The reagents;        -   (a) bind specifically to, or react specifically with, the            product, and not the substrate, if both substrate and            product are attached to the genetic element, or        -   (b) optionally bind both substrate and product if only the            product, and not the substrate binds to, or reacts with, the            genetic element.

The pooled genetic elements encoding catalytic molecules can then beenriched by selecting for the genetic elements with modified opticalproperties.

An alternative is to couple the nucleic acid to a product-specificantibody (or other product-specific molecule). In this scenario, thesubstrate (or one of the substrates) is present in each microcapsuleunlinked to the genetic element, but has a molecular “tag” (for examplebiotin, DIG or DNP or a fluorescent group). When the catalyst to beselected converts the substrate to product, the product retains the“tag” and is then captured in the microcapsule by the product-specificantibody. In this way the genetic element only becomes associated withthe “tag” when it encodes or produces an enzyme capable of convertingsubstrate to product. When all reactions are stopped and themicrocapsules are combined, the genetic elements encoding active enzymeswill be “tagged” and may already have changed optical properties, forexample, if the “tag” was a fluorescent group. Alternatively, a changein optical properties of “tagged” genes can be induced by adding afluorescently labelled ligand which binds the “tag” (for examplefluorescently-labelled avidin/streptavidin, an anti-“tag” antibody whichis fluorescent, or a non-fluorescent anti-“tag” antibody which can bedetected by a second fluorescently-labelled antibody).

Alternatively, selection may be performed indirectly by coupling a firstreaction to subsequent reactions that takes place in the samemicrocapsule. There are two general ways in which this may be performed.In a first embodiment, the product of the first reaction is reactedwith, or bound by, a molecule which does not react with the substrate ofthe first reaction. A second, coupled reaction will only proceed in thepresence of the product of the first reaction. A genetic elementencoding a gene product with a desired activity can then be purified byusing the properties of the product of the second reaction to induce achange in the optical properties of the genetic element as above.

Alternatively, the product of the reaction being selected may be thesubstrate or cofactor for a second enzyme-catalysed reaction. The enzymeto catalyse the second reaction can either be translated in situ in themicrocapsules or incorporated in the reaction mixture prior tomicroencapsulation. Only when the first reaction proceeds will thecoupled enzyme generate a product which can be used to induce a changein the optical properties of the genetic element as above.

This concept of coupling can be elaborated to incorporate multipleenzymes, each using as a substrate the product of the previous reaction.This allows for selection of enzymes that will not react with animmobilised substrate. It can also be designed to give increasedsensitivity by signal amplification if a product of one reaction is acatalyst or a cofactor for a second reaction or series of reactionsleading to a selectable product (for example, see Johannsson and Bates,1988; Johannsson, 1991). Furthermore an enzyme cascade system can bebased on the production of an activator for an enzyme or the destructionof an enzyme inhibitor (see Mize et al., 1989). Coupling also has theadvantage that a common selection system can be used for a whole groupof enzymes which generate the same product and allows for the selectionof complicated chemical transformations that cannot be performed in asingle step.

Such a method of coupling thus enables the evolution of novel “metabolicpathways” in vitro in a stepwise fashion, selecting and improving firstone step and then the next. The selection strategy is based on the finalproduct of the pathway, so that all earlier steps can be evolvedindependently or sequentially without setting up a new selection systemfor each step of the reaction.

Expressed in an alternative manner, there is provided a method ofisolating one or more genetic elements encoding a gene product having adesired catalytic activity, comprising the steps of:

-   -   (1) expressing genetic elements to give their respective gene        products;    -   (2) allowing the gene products to catalyse conversion of a        substrate to a product, which may or may not be directly        selectable, in accordance with the desired activity;    -   (3) optionally coupling the first reaction to one or more        subsequent reactions, each reaction being modulated by the        product of the previous reactions, and leading to the creation        of a final, selectable product;    -   (4) linking the selectable product of catalysis to the genetic        elements by either:        -   a) coupling a substrate to the genetic elements in such a            way that the product remains associated with the genetic            elements, or        -   b) reacting or binding the selectable product to the genetic            elements by way of a suitable molecular “tag” attached to            the substrate which remains on the product,        -   or        -   c) coupling the selectable product (but not the substrate)            to the genetic elements by means of a product-specific            reaction or interaction with the product; and    -   (5) selecting the product of catalysis, together with the        genetic element to which it is bound, either by means of its        characteristic optical properties, or by adding reagents which        specifically bind to, or react specifically with, the product        and which thereby induce a change in the optical properties of        the genetic elements wherein steps (1) to    -   (6) each genetic element and respective gene product is        contained within a microcapsule.        (iii) Selecting for Enzyme Substrate Specificity/Selectivity

Genetic elements encoding enzymes with substrate specificity orselectivity can be specifically enriched by carrying out a positiveselection for reaction with one substrate and a negative selection forreaction with another substrate. Such combined positive and negativeselection pressure should be of great importance in isolatingregio-selective and stereo-selective enzymes (for example, enzymes thatcan distinguish between two enantiomers of the same substrate). Forexample, two substrates (e.g. two different enantiomers) are eachlabelled with different tags (e.g. two different fluorophores) such thatthe tags become attached to the genetic element by the enzyme-catalysedreaction. If the two tags confer different optical properties on thegenetic element the substrate specificity of the enzyme can bedetermined from the optical properties of the genetic element and thosegenetic elements encoding gene products with the wrong (or no)specificity rejected. Tags conferring no change in optical activity canalso be used if tag-specific ligands with different optical propertiesare added (e.g. tag-specific antibodies labelled with differentfluorophores).

(iv) Selection for Regulation

A similar system can be used to select for regulatory properties ofenzymes.

In the case of selection for a regulator molecule which acts as anactivator or inhibitor of a biochemical process, the components of thebiochemical process can either be translated in situ in eachmicrocapsule or can be incorporated in the reaction mixture prior tomicroencapsulation. If the genetic element being selected is to encodean activator, selection can be performed for the product of theregulated reaction, as described above in connection with catalysis. Ifan inhibitor is desired, selection can be for a chemical propertyspecific to the substrate of the regulated reaction.

There is therefore provided a method of sorting one or more geneticelements coding for a gene product exhibiting a desired regulatoryactivity, comprising the steps of:

-   -   (1) expressing genetic elements to give their respective gene        products;    -   (2) allowing the gene products to activate or inhibit a        biochemical reaction, or sequence of coupled reactions, in        accordance with the desired activity, in such a way as to allow        the generation or survival of a selectable molecule;    -   (3) linking the selectable molecule to the genetic elements        either by        -   a) having the selectable molecule, or the substrate from            which it derives, attached to the genetic elements, or        -   b) reacting or binding the selectable product to the genetic            elements, by way of a suitable molecular “tag” attached to            the substrate which remains on the product,        -   or        -   c) coupling the product of catalysis (but not the substrate)            to the genetic elements, by means of a product-specific            reaction or interaction with the product;    -   (4) selecting the selectable product, together with the genetic        element to which it is bound, either by means of its        characteristic optical properties, or by adding reagents which        specifically bind to, or react specifically with, the product        and which thereby induce a change in the optical properties of        the genetic elements wherein steps (1) to (3) each genetic        element and respective gene product is contained within a        microcapsule.

(v) Selection for Optical Properties of the Gene Product

It is possible to select for inherent optical properties of geneproducts if, in the microcapsules, the gene product binds back to thegenetic element, for example through a common element of the geneproduct which binds to a ligand which is part of the genetic element.After pooling the genetic elements they can then be sorted using theoptical properties of the bound gene products. This embodiment can beused, for example, to select variants of green fluorescent protein (GFP)(Cormack et al., 1996; Delagrave et al., 1995; Ehrig et al., 1995), withimproved fluorescence and/or novel absoption and emission spectra.

(vi) Screening Using Cells

In the current drug discovery paradigm, validated recombinant targetsform the basis of in vitro high-throughput screening (HTS) assays.Isolated genetic constructs or polypeptides cannot, however, be regardedas representative of complex biological systems; hence, cell-basedsystems can provide greater confidence in compound activity in an intactbiological system. A wide range of cell-based assays for drug leads areknown to those skilled in the art. Cells can be compartmentalised inmicrocapsules, such as the aqueous microdroplets of a water-in-oilemulsion (Ghadessy, 2001). The effect of a compound(s) on a target canbe determined by compartmentalising a cell (or cells) in a microcapsuletogether with a genetic element(s) and using an appropriate cell-basedassay to identify those compartments containing genetic elements withthe desired effect on the cell(s). The use of water-in-fluorocarbonemulsions may be particularly advantageous: the high gas dissolvingcapacity of fluorocarbons can support the exchange of respiratory gasesand has been reported to be beneficial to cell culture systems (Lowe,2002).

(vii) Flow Analysis and Sorting

In a preferred embodiment of the invention the microcapsules will beanalysed and, optionally, sorted by flow cytometry. Many formats ofmicrocapsule can be analysed and, optionally, sorted directly using flowcytometry.

In a highly preferred embodiment, microfluidic devices for flow analysisand, optionally, flow sorting (Fu, 2002) of microcapsules will be used.Such a sorting device can be integrated directly on the microfluidicdevice, and can use electronic means to sort the microcapsules and/orgenetic elements. Optical detection, also integrated directly on themicrofluidic device, can be used to screen the microcapsules to triggerthe sorting. Other means of control of the microcapsules, in addition tocharge, can also be incorporated onto the microfluidic device.

A variety of optical properties can be used for analysis and to triggersorting, including light scattering (Kerker, 1983) and fluorescencepolarisation (Rolland et al., 1985). In a highly preferred embodimentthe difference in optical properties of the microcapsules or microbeadswill be a difference in fluorescence and, if required, the microcapsulesor microbeads will be sorted using a microfluidic or conventionalfluorescence activated cell sorter (Norman, 1980; Mackenzie and Pinder,1986), or similar device. Flow cytometry has a series of advantages:

-   -   (1) fluorescence activated cell sorting equipment from        established manufacturers (e.g. Becton-Dickinson, Coulter,        Cytomation) allows the analysis and sorting at up to 100,000        microcapsules or microbeads per second.    -   (2) the fluorescence signal from each microcapsule or microbead        corresponds tightly to the number of fluorescent molecules        present. As little as few hundred fluorescent molecules per        microcapsules or microbeads can be quantitatively detected;    -   (3) the wide dynamic range of the fluorescence detectors        (typically 4 log units) allows easy setting of the stringency of        the sorting procedure, thus allowing the recovery of the optimal        number microcapsules or microbeads from the starting pool (the        gates can be set to separate microcapsules or microbeads with        small differences in fluorescence or to only separate out        microcapsules or microbeads with large differences in        fluorescence, dependant on the selection being performed);    -   (4) fluorescence-activated cell sorting equipment can perform        simultaneous excitation and detection at multiple wavelengths        (Shapiro, 1995) allowing positive and negative selections to be        performed simultaneously by monitoring the labelling of the        microcapsules or microbeads with two to thirteen (or more)        fluorescent markers, for example, if substrates for two        alternative targets are labelled with different fluorescent tags        the microcapsules or microbeads can labelled with different        fluorophores dependent on the target regulated.

If the microcapsules or microbeads are optically tagged, flow cytometrycan also be used to identify the genetic element or genetic elements inthe microcapsule or coated on the microbeads (see below). Opticaltagging can also be used to identify the concentration of reagents inthe microcapsule (if more than one concentration is used in a singleexperiment) or the number of compound molecules coated on a microbead(if more than one coating density is used in a single experiment).Furthermore, optical tagging can be used to identify the target in amicrocapsule (if more than one target is used in a single experiment).This analysis can be performed simultaneously with measuring activity,after sorting of microcapsules containing microbeads, or after sortingof the microbeads.

(viii) Microcapsule Identification and Sorting

The invention provides for the identification and, optionally, thesorting of intact microcapsules where this is enabled by the sortingtechniques being employed. Microcapsules may be identified and,optionally, sorted as such when the change induced by the desiredgenetic element either occurs or manifests itself at the surface of themicrocapsule or is detectable from outside the microcapsule. The changemay be caused by the direct action of the gene product, or indirect, inwhich a series of reactions, one or more of which involve the geneproduct having the desired activity leads to the change. For example,where the microcapsule is a membranous microcapsule, the microcapsulemay be so configured that a component or components of the biochemicalsystem comprising the target are displayed at its surface and thusaccessible to reagents which can detect changes in the biochemicalsystem regulated by the gene product within the microcapsule.

In a preferred aspect of the invention, however, microcapsuleidentification and, optionally, sorting relies on a change in theoptical properties of the microcapsule, for example absorption oremission characteristics thereof, for example alteration in the opticalproperties of the microcapsule resulting from a reaction leading tochanges in absorbance, luminescence, phosphorescence or fluorescenceassociated with the microcapsule. All such preperties are included inthe term “optical”. In such a case, microcapsules can be identified and,optionally, sorted by luminescence, fluorescence or phosphorescenceactivated sorting. In a highly preferred embodiment, flow cytometry isemployed to analyse and, optionally, sort microcapsules containing geneproducts having a desired activity which result in the production of afluorescent molecule in the microcapsule.

The methods of the current invention allow reagents to be mixed rapidly(in <2 ms), hence a spatially-resolved optical image of microcapsules inmicrofluidic network allows time resolved measurements of the reactionsin each microcapsule. Microcapsules can, optionally, be separated usinga microfluidic flow sorter to allow recovery and further analysis ormanipulation of the molecules they contain. Advantageously, the flowsorter would be an electronic flow sorting device. Such a sorting devicecan be integrated directly on the microfluidic device, and can useelectronic means to sort the microcapsules. Optical detection, alsointegrated directly on the microfluidic device, can be used to screenthe microcapsules to trigger the sorting. Other means of control of themicrocapsules, in addition to charge, can also be incorporated onto themicrofluidic device.

In an alternative embodiment, a change in microcapsule fluorescence,when identified, is used to trigger the modification of the microbeadwithin the compartment. In a preferred aspect of the invention,microcapsule identification relies on a change in the optical propertiesof the microcapsule resulting from a reaction leading to luminescence,phosphorescence or fluorescence within the microcapsule. Modification ofthe microbead within the microcapsules would be triggered byidentification of luminescence, phosphorescence or fluorescence. Forexample, identification of luminescence, phosphorescence or fluorescencecan trigger bombardment of the compartment with photons (or otherparticles or waves) which leads to modification of the microbead ormolecules attached to it. A similar procedure has been describedpreviously for the rapid sorting of cells (Keij et al., 1994).Modification of the microbead may result, for example, from coupling amolecular “tag”, caged by a photolabile protecting group to themicrobeads: bombardment with photons of an appropriate wavelength leadsto the removal of the cage. Afterwards, all microcapsules arc combinedand the microbeads pooled together in one environment. Genetic elementsexhibiting the desired activity can be selected by affinity purificationusing a molecule that specifically binds to, or reacts specificallywith, the “tag”.

(ix) Flow Sorting of Genetic Elements

In a preferred embodiment of the invention the genetic elements will besorted by flow cytometry. A variety of optical properties can be used totrigger sorting, including light scattering (Kerker, 1983) andfluorescence polarisation (Rolland et al., 1985). In a highly preferredembodiment the difference in optical properties of the genetic elementswill be a difference in fluorescence and the genetic elements will besorted using a fluorescence activated cell sorter (Norman, 1980;Mackenzie and Pinder, 1986), or similar device. Such a sorting devicecan be integrated directly on the microfluidic device, and can useelectronic means to sort the genetic elements. Optical detection, alsointegrated directly on the microfluidic device, can be used to screenthe genetic elements to trigger the sorting. Other means of control ofthe genetic elements, in addition to charge, can also be incorporatedonto the microfluidic device. In an especially preferred embodiment thegenetic element comprises of a nonfluorescent nonmagnetic (e.g.polystyrene) or paramagnetic microbead (see Formusek and Vetvicka,1986), optimally 0.6 to 1.0 μm diameter, to which are attached both thegene and the groups involved in generating a fluorescent signal:

-   -   (1) commercially available fluorescence activated cell sorting        equipment from established manufacturers (e.g. Becton-Dickinson,        Coulter) allows the sorting of up to 10⁸ genetic elements        (events) per hour;    -   (2) the fluorescence signal from each bead corresponds tightly        to the number of fluorescent molecules attached to the bead. At        present as little as few hundred fluorescent molecules per        particle can be quantitatively detected;    -   (3) the wide dynamic range of the fluorescence detectors        (typically 4 log units) allows easy setting of the stringeney of        the sorting procedure, thus allowing the recovery of the optimal        number of genetic elements from the starting pool (the gates can        be set to separate beads with small differences in fluorescence        or to only separate out beads with large differences in        fluorescence, dependant on the selection being performed;    -   (4) commercially available fluorescence-activated cell sorting        equipment can perform simultaneous excitation at up to two        different wavelengths and detect fluoreseence at up to four        different wavelengths (Shapiro, 1983) allowing positive and        negative selections to be performed simultaneously by monitoring        the labelling of the genetic element with two (or more)        different fluorescent markers, for example, if two alternative        substrates for an enzyme (e.g. two different enantiomers) are        labelled with different fluorescent tags the genetic element can        labelled with different fluorophores dependent on the substrate        used and only genes encoding enzymes with enantioselectivity        selected.    -   (5) highly uniform derivatised and non-derivatised nonmagnetic        and paramagnetic microparticles (beads) are commercially        available from many sources (e.g. Sigma, and Molecular Probes)        (Formusek and Vetvicka, 1986).

(x) Multi-Step Procedure

It will be also be appreciated that according to the present invention,it is not necessary for all the processes of transcription/replicationand/or translation, and selection to proceed in one single step, withall reactions taking place in one microcapsule. The selection proceduremay comprise two or more steps. First, transcription/replication and/ortranslation of each genetic element of a genetic element library maytake place in a first microcapsule. Each gene product is then linked tothe genetic element which encoded it (which resides in the samemicrocapsule), for example via a gene product-specific ligand such as anantibody. The microcapsules are then broken, and the genetic elementsattached to their respective gene products optionally purified.Alternatively, genetic elements can be attached to their respective geneproducts using methods which do not rely on encapsulation. For examplephage display (Smith, G. P., 1985), polysome display (Mattheakkis etal., 1994), RNA-peptide fusion (Roberts and Szostak, 1997) or lacrepressor peptide fusion (Cull, et al., 1992).

In the second step of the procedure, each purified genetic elementattached to its gene product is put into a second microcapsulecontaining components of the reaction to be selected. This reaction isthen initiated. After completion of the reactions, the microcapsules areagain broken and the modified genetic elements are selected. In the caseof complicated multistep reactions in which many individual componentsand reaction steps are involved, one or more intervening steps may beperformed between the initial step of creation and linking of geneproduct to genetic element, and the final step of generating theselectable change in the genetic element.

If necessary, release of the gene product from the genetic elementwithin a secondary microcapsule can be achieved in a variety of ways,including by specific competition by a low-molecular weight product forthe binding site or cleavage of a linker region joining the bindingdomain of the gene product from the catalytic domain eitherenzymatically (using specific proteases) or autocatalytically (using anintegrin domain).

(xi) Selection by Activation of Reporter Gene Expression in Situ

The system can be configured such that the desired binding, catalytic orregulatory activity encoded by a genetic element leads, directly orindirectly to the activation of expression of a “reporter gene” that ispresent in all microcapsules. Only gene products with the desiredactivity activate expression of the reporter gene. The activityresulting from reporter gene expression allows the selection of thegenetic element (or of the compartment containing it) by any of themethods described herein.

For example, activation of the reporter gene may be the result of abinding activity of the gene product in a manner analogous to the “twohybrid system” (Fields and Song; 1989). Activation can also result fromthe product of a reaction catalysed by a desirable gene product. Forexample, the reaction product can be a transcriptional inducer of thereporter gene. For example arabinose may be used to induce transcriptionfrom the araBAD promoter. The activity of the desirable gene product canalso result in the modification of a transcription factor, resulting inexpression of the reporter gene. For example, if the desired geneproduct is a kinase or phosphatase the phosphorylation ordephosphorylation of a transcription factor may lead to activation ofreporter gene expression.

(xii) Amplification

According to a further aspect of the present invention the methodcomprises the further step of amplifying the genetic elements. Selectiveamplification may be used as a means to enrich for genetic elementsencoding the desired gene product.

In all the above configurations, genetic material comprised in thegenetic elements may be amplified and the process repeated in iterativesteps. Amplification may be by the polymerase chain reaction (Saiki etal., 1988) or by using one of a variety of other gene amplificationtechniques including; Qb replicase amplification (Cahill, Foster andMahan, 1991; Chetverin and Spirin, 1995; Katanaev, Kurnasov and Spirin,1995); the ligase chain reaction (LCR) (Landegren et al., 1988; Barany,1991); the self-sustained sequence replication system (Fahy, Kwoh andGingeras, 1991) and strand displacement amplification (Walker et al.,1992). Advantageously, the amplification procedure can be performed in amicrofluidic device.

(C) Rapid Mixing of Reagents in Microcapsules

Advantageously, after fusion of microcapsules, the reagents contained inthe fused microcapsule can be mixed rapidly using chaotic advection bypassing the droplets through channels that disrupt the laminar flowlines of the fluid within the droplets, their contents can be rapidlymixed, fully initiating any chemical reactions.

(D) Sensing Microcapsule Characteristics

In certain aspects of the invention, sensors are provided that can senseand/or determine one or more characteristics of the fluidic droplets,and/or a characteristic of a portion of the fluidic system containingthe fluidic droplet (e.g., the liquid surrounding the fluidic droplet)in such a manner as to allow the determination of one or morecharacteristics of the fluidic droplets. Characteristics determinablewith respect to the droplet and usable in the invention can beidentified by those of ordinary skill in the art. Non-limiting examplesof such characteristics include fluorescence, spectroscopy (e.g.,optical, infrared, ultraviolet, etc.), radioactivity, mass, volume,density, temperature, viscosity, pH, concentration of a substance, suchas a biological substance (e.g., a protein, a nucleic acid, etc.), orthe like.

In some cases, the sensor may be connected to a processor, which inturn, causes an operation to be performed on the fluidic droplet, forexample, by sorting the droplet, adding or removing electric charge fromthe droplet, fusing the droplet with another droplet, splitting thedroplet, causing mixing to occur within the droplet, etc., for example,as previously described. For instance, in response to a sensormeasurement of a fluidic droplet, a processor may cause the fluidicdroplet to be split, merged with a second fluidic droplet, sorted etc.

One or more sensors and/or processors may be positioned to be in sensingcommunication with the fluidic droplet. “Sensing communication,” as usedherein, means that the sensor may be positioned anywhere such that thefluidic droplet within the fluidic system (e.g., within a channel),and/or a portion of the fluidic system containing the fluidic dropletmay be sensed and/or determined in some fashion. For example, the sensormay be in sensing communication with the fluidic droplet and/or theportion of the fluidic system containing the fluidic droplet fluidly,optically or visually, thermally, pneumatically, electronically, or thelike. The sensor can be positioned proximate the fluidic system, forexample, embedded within or integrally connected to a wall of a channel,or positioned separately from the fluidic system but with physical,electrical, and/or optical communication with the fluidic system so asto be able to sense and/or determine the fluidic droplet and/or aportion of the fluidic system containing the fluidic droplet (e.g., achannel or a microchannel, a liquid containing the fluidic droplet,etc.). For example, a sensor may be free of any physical connection witha channel containing a droplet, but may be positioned so as to detectelectromagnetic radiation arising from the droplet or the fluidicsystem, such as infrared, ultraviolet, or visible light. Theelectromagnetic radiation may be produced by the droplet, and/or mayarise from other portions of the fluidic system (or externally of thefluidic system) and interact with the fluidic droplet and/or the portionof the fluidic system containing the fluidic droplet in such as a manneras to indicate one or more characteristics of the fluidic droplet, forexample, through absorption, reflection, diffraction, refraction,fluorescence, phosphorescence, changes in polarity, phase changes,changes with respect to time, etc. As an example, a laser may bedirected towards the fluidic droplet and/or the liquid surrounding thefluidic droplet, and the fluorescence of the fluidic droplet and/or thesurrounding liquid may be determined. “Sensing communication,” as usedherein may also be direct or indirect. As an example, light from thefluidic droplet may be directed to a sensor, or directed first through afiber optic system, a waveguide, etc., before being directed to asensor.

Non-limiting examples of sensors useful in the invention include opticalor electromagnetically-based systems. For example, the sensor may be afluorescence sensor (e.g., stimulated by a laser), a microscopy system(which may include a camera or other recording device), or the like. Asanother example, the sensor may be an electronic sensor, e.g., a sensorable to determine an electric field or other electrical characteristic.For example, the sensor may detect capacitance, inductance, etc., of afluidic droplet and/or the portion of the fluidic system containing thefluidic droplet.

As used herein, a “processor” or a “microprocessor” is any component ordevice able to receive a signal from one or more sensors, store thesignal, and/or direct one or more responses (e.g., as described above),for example, by using a mathematical formula or an electronic orcomputational circuit. The signal may be any suitable signal indicativeof the environmental factor determined by the sensor, for example apneumatic signal, an electronic signal, an optical signal, a mechanicalsignal, etc.

As a particular non-limiting example, a device of the invention maycontain fluidic droplets containing one or more cells. The desiredactivity of one or more gene products may result in the expression (orinhibition of expression) of a ‘marker’ gene, for example a gene forgreen fluorescent protein (GFP). The cells may be exposed to afluorescent signal marker that binds if a certain condition is present,for example, the marker may bind to a first cell type but not a secondcell type, the marker may bind to an expressed protein, the marker mayindicate viability of the cell (i.e., if the cell is alive or dead), themarker may be indicative of the state of development or differentiationof the cell, etc., and the cells may be directed through a fluidicsystem of the invention based on the presence/absence, and/or magnitudeof the fluorescent signal marker. For instance, determination of thefluorescent signal marker may cause the cells to be directed to oneregion of the device (e.g., a collection chamber), while the absence ofthe fluorescent signal marker may cause the cells to be directed toanother region of the device (e.g., a waste chamber). Thus, in thisexample, a population of cells may be screened and/or sorted on thebasis of one or more determinable or targetable characteristics of thecells, for example, to select live cells, cells expressing a certainprotein, a certain cell type, etc.

(E) Materials

A variety of materials and methods, according to certain aspects of theinvention, can be used to form any of the above-described components ofthe microfluidic systems and devices of the invention. In some cases,the various materials selected lend themselves to various methods. Forexample, various components of the invention can be formed from solidmaterials, in which the channels can be formed via micromachining, filmdeposition processes such as spin coating and chemical vapor deposition,laser fabrication, photolithographic techniques, etching methodsincluding wet chemical or plasma processes, and the like. See, forexample, Scientific American, 248:44-55, 1983 (Angell, et al). In oneembodiment, at least a portion of the fluidic system is formed ofsilicon by etching features in a silicon chip. Technologies for preciseand efficient fabrication of various fluidic systems and devices of theinvention from silicon are known. In another embodiment, variouscomponents of the systems and devices of the invention can be formed ofa polymer, for example, an elastomeric polymer such aspolydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE” orTeflon®), or the like.

Different components can be fabricated of different materials. Forexample, a base portion including a bottom wall and side walls can befabricated from an opaque material such as silicon or PDMS, and a topportion can be fabricated from a transparent or at least partiallytransparent material, such as glass or a transparent polymer, forobservation and/or control of the fluidic process. Components can becoated so as to expose a desired chemical functionality to fluids thatcontact interior channel walls, where the base supporting material doesnot have a precise, desired functionality. For example, components canbe fabricated as illustrated, with interior channel walls coated withanother material. Material used to fabricate various components of thesystems and devices of the invention, e.g., materials used to coatinterior walls of fluid channels, may desirably be selected from amongthose materials that will not adversely affect or be affected by fluidflowing through the fluidic system, e.g., material(s) that is chemicallyinert in the presence of fluids to be used within the device.

In one embodiment, various components of the invention are fabricatedfrom polymeric and/or flexible and/or elastomeric materials, and can beconveniently formed of a hardenable fluid, facilitating fabrication viamolding (e.g. replica molding, injection molding, cast molding, etc.).The hardenable fluid can be essentially any fluid that can be induced tosolidify, or that spontaneously solidifies, into a solid capable ofcontaining and/or transporting fluids contemplated for use in and withthe fluidic network. In one embodiment, the hardenable fluid comprises apolymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).Suitable polymeric liquids can include, for example, thermoplasticpolymers, thermoset polymers, or mixture of such polymers heated abovetheir melting point. As another example, a suitable polymeric liquid mayinclude a solution of one or more polymers in a suitable solvent, whichsolution forms a solid polymeric material upon removal of the solvent,for example, by evaporation. Such polymeric naaterials, which can besolidified from, for example, a melt state or by solvent evaporation,are well known to those of ordinary skill in the art. A variety ofpolymeric materials, many of which are elastomeric, are suitable, andare also suitable for forming molds or mold masters, for embodimentswhere one or both of the mold masters is composed of an elastomericmaterial. A non-limiting list of examples of such polymers includespolymers of the general classes of silicone polymers, epoxy polymers,and acrylate polymers. Epoxy polymers are characterized by the presenceof a three-membered cyclic ether group commonly referred to as an epoxygroup, 1,2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Non-limiting examples of siliconeelastomers suitable for use according to the invention include thoseformed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred in one set of embodiments, for example,the silicone elastomer polydimethylsiloxane. Non-limiting examples ofPDMS polymers include those sold under the trademark Sylgard by DowChemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184,and Sylgard 186. Silicone polymers including PDMS have severalbeneficial properties simplifying fabrication of the microfluidicstructures of the invention. For instance, such materials areinexpensive, readily available, and can be solidified from aprepolymeric liquid via curing with heat. For example, PDMSs aretypically curable by exposure of the prepolymeric liquid to temperaturesof about, for example, about 65° C. to about 75° C. for exposure timesof, for example, about an hour. Also, silicone polymers, such as PDMS,can be elastomeric and thus may be useful for forming very smallfeatures with relatively high aspect ratios, necessary in certainembodiments of the invention. Flexible (e.g., elastomeric) molds ormasters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus, components can be fabricated and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention, as well as overall molding techniques, are described in theart, for example, in an article entitled “Rapid Prototyping ofMicrofluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480,1998 (Duffy et al.), incorporated herein by reference.

Another advantage to forming microfluidic structures of the invention(or interior, fluid contacting surfaces) from oxidized silicone polymersis that these surfaces can be much more hydrophilic than the surfaces oftypical elastomeric polymers (where a hydrophilic interior surface isdesired). Such hydrophilic channel surfaces can thus be more easilyfilled and wetted with aqueous solutions than can structures comprisedof typical, unoxidized elastomeric polymers or other hydrophobicmaterials.

In one embodiment, a bottom wall is formed of a material different fromone or more side walls or a top wall, or other components. For example,the interior surface of a bottom wall can comprise the surface of asilicon wafer or microchip, or other substrate. Other components can, asdescribed above, be sealed to such alternative substrates. Where it isdesired to seal a component comprising a silicone polymer (e.g. PDMS) toa substrate (bottom wall) of different material, the substrate may beselected from the group of materials to which oxidized silicone polymeris able to irreversibly seal (e.g., glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, andglassy carbon surfaces which have been oxidized). Alternatively, othersealing techniques can be used, as would be apparent to those ofordinary skill in the art, including, but not limited to, the use ofseparate adhesives, thermal bonding, solvent bonding, ultrasonicwelding, etc.

Various aspects and embodiments of the present invention are illustratedin the following examples. It will be appreciated that modification ofdetail may be made without departing from the scope of the invention.

All documents mentioned in the text are incorporated by reference.

EXAMPLES Example 1 Microfluidic Device for Selection of Genes Using InVitro Compartmentalisation

A schematic representation of the microfluidic device is shown in FIG.15. Microchannels are fabricated with rectangular cross-sections usingrapid prototyping in poly(dimethylsiloxane) (PDMS) (McDonald andWhitesides, 2002) and rendered hydrophobic as (Song and Ismagilov,2003). Syringe pumps were used to drive flows (Harvard Apparatus PHD2000 Infusion pumps). For aqueous solutions, 250 μi Hamilton Gastightsyringes (1700 series, TLL) with removeable needles of 27-gaugeare usedwith 30-gauge Teflon tubing (WeiCo Wire and Cable). For the carrierfluid, 1 ml Hamilton Gastight syringes (1700 series, TLL) are used with30-gauge Teflon needles with one hub from Hamilton (Song and Ismagilov,2003). The carrier fluid is 9% (v/v) C₆F₁₁C₂H₄OH in perfluorodecaline(PFD) (Song et al., 2003). The microfluidic device consists of a seriesof interconnected modules. Each module has a specific function. Theseinclude modules that will produce droplets, fuse droplets, mix droplets,react droplets, detect droplets, and 20 sort droplets (see FIG. 16). Inone example, droplets are made, consisting of different molecules ordifferent concentrations of molecules. Droplets are made at rates of upto 10⁴ sec⁻¹, and are made with a polydispersity of less than 1.5% andwith sizes ranging from 1 μm to 100 μm. Each droplet is fused with asecond droplet containing a second set of reactants, and is rapidlymixed to initiate the chemical reaction. This chemical reaction isallowed to proceed in each droplet by passing it through a delaychannel. Each droplet is then fused with another droplet containing asecond set of reactants, and is subsequently rapidly mixed to initiatethe second set of chemical reactions. After the second reaction hasproceeded in a delay module, the results of the reaction is determinedusing an optical sensor or other form of detection module. Finally, thedesired droplets are sorted into two populations based on signal formthe optical detection module, one population is kept for furtherprocessing and the other discarded. These and other modules can be usedin this combination, or in other combinations.

Droplet Generation Module:

We use a flow-focusing geometry to form the drops. A water stream isinfused from one channel through a narrow constriction; counterpropagating oil streams hydrodynamically focus the water stream reducingits size as it passes through the constriction as shown in FIG. 17A.This droplet generator can be operated in a flow regime that produces asteady stream of uniform droplets of water in oil. The size of the waterdroplets is controlled by the relative flow rates of the oil and thewater; the viscous forces overcome surface tension to create uniformdroplets. If the flow rate of the water is too high a longer jet offluid passes through the orifice and breaks up into droplets furtherdown stream; these droplets are less uniform in size. If the flow rateof the water is too low, the droplet breakup in the orifice becomesirregular again, producing a wider range of droplet sizes. While thisemulsification technology is robust, it is limited to producing dropletsof one size at any given flow rate; this droplet size is largelydetermined by the channel dimensions. Moreover, the timing of thedroplet production cannot be controlled.

We overcome these limitations by incorporating electric fields to createan electrically addressable emulsification system. To achieve this, weapply high voltage to the aqueous stream and charge the oil waterinterface, as shown schematically in FIG. 17A. The water stream behavesas a conductor while the oil is an insulator; electrochemical reactionscharge the fluid interface like a capacitor. At snap-off, charge on theinterface remains on the droplet. In addition, the droplet volume,V_(d), and frequency, f, can be tailored over nearly three orders ofmagnitude without changing the infusion rate of the oil or water.Droplet size and frequency are not independent; instead their product isdetermined by the infusion rate of the dispersed phase Q_(d)=fV_(d). Thedroplet size decreases with increasing field strength, as shown in FIG.17, B to E. The dependence of the droplet size on applied voltage forthree different flow rates is summarized in FIG. 17F. At low appliedvoltages the electric field has a negligible effect, and dropletformation is driven exclusively by the competition between surfacetension and viscous flow. By contrast, at high electric field strengths,there is a significant additional force on the growing drop, F=qE, whereq is the charge on the droplet. Since the droplet interface behaves as acapacitor, q is proportional to the applied voltage, V. This leads to aV² dependence of the force, which accounts for the decrease in dropletsize with increasing applied field shown in FIG. 17F. If the electricfield becomes too large, the charged interface of the water stream isrepelled by the highly charged drops; this destabilizes the productionand increases the variation in droplet size.

The electronic control afforded by the field-induced droplet formationprovides an additional valuable benefit: it allows the phase of thedroplet break-off to be adjusted within the production cycle. This isaccomplished by increasing the field above the critical break-off fieldonly at the instant the droplet is required. This provides a convenientmeans to precisely synchronize the production and arrival of individualdroplets at specific locations.

Droplet Coalesces Module:

An essential component in any droplet-based reaction confinement systemis a droplet coalescing module which combines two or more reagents toinitiate a chemical reaction. This is particularly difficult to achievein a microfluidic device because surface tension, surfactantstabilization, and drainage forces all hinder droplet coalescence;moreover, the droplets must cross the stream lines that define theirrespective flows and must be perfectly synchronized to arrive at aprecise location for coalescence.

Use of electrostatic charge overcomes these difficulties; placingcharges of opposite sign on each droplet and applying an electric fieldforces them to coalesce. As an example we show a device consisting oftwo separate nozzles that generate droplets with different compositionsand opposite charges, sketched in FIG. 18A. The droplets are broughttogether at the confluence of the two streams. The electrodes used tocharge the droplets upon formation also provide the electric field toforce the droplets across the stream lines, leading to coalesce. Slightvariations in the structure of the two nozzles result in slightdifferences in the frequency and phase of their droplet generation inthe absence of a field. Thus the droplets differ in size even though theinfusion rates are identical. Moreover, the droplets do not arrive atthe point of confluence at exactly the same time. As a result thedroplets do not coalesce as shown in FIG. 18B. By contrast, uponapplication of an electric field, droplet formation becomes exactlysynchronized, ensuring that pairs of identically sized droplets eachreach the point of confluence simultaneously. Moreover, the droplets areoppositely charged, forcing them to traverse the stream lines andcontact each other, thereby causing them to coalesce, as shown in FIG.18C. The remarkable synchronization of the droplet formation resultsfrom coupling of the break-off of each of the pair of droplets asmediated by the electric field; the magnitude of the electric fieldvaries as the separation between the leading edges of the two dropletschanges and the frequency of droplet break-off is mode-locked to theelectric field. A minimum charge is required to cause droplets tocoalesce, presumably because of the stabilizing effects of thesurfactant coating; this is clear from FIG. 18D which shows the voltagedependence of the percentage of drops that contact each other thatactually coalesce.

Droplet Mixer Module:

Rapid mixing is achieved through either successive iterations oftranslation and rotation, FIG. 19, or by coalescing drops along thedirection parallel to the flow direction, FIG. 20.

Droplet Reactor/Time Delay Module:

A delay line is used to provide a fixed time for a reaction. Twonon-limiting examples of how this can be achieved are ‘single file’ and‘large cross-section’ channels. The ‘single file’ delay line uses lengthto achieve a fixed reaction time. As this often results in exceptionallylong channels, it is desirable to place spacer droplets of a thirdfluid, immicible with both the carrier oil and the aqueous dropletsinbetween aqueous droplet pairs. There is then an alternation betweenaqueous and non-aqueous droplets in a carrier oil. This is shown in FIG.21A. A second possibility for achieving a long time delay is to use wideand deap channel having a ‘large cross-sectiononal area’ to slow theaverage velocity of the droplets. An example of this is shown in FIG.21B.

Recharging Module:

The use of oppositely charged droplets and an electric field to combineand mix reagents is extremely robust, and 100% of the droplets coalescewith their partner from the opposite stream. However, after theycoalesce the resultant drops carry no electrostatic charge. While it isconvenient to charge droplets during formation, other methods must beemployed in any robust droplet-based micro fluidic system to rechargethe mixed droplets if necessary for further processing. This is readilyaccomplished through the use of extensional flow to split neutraldroplets in the presence of an electric field which polarizes them,resulting in two oppositely charged daughter droplets; this is sketchedin FIG. 22A. The photomicrograph in FIG. 22B shows neutral dropletsentering a bifurcation and splitting into charged daughter droplets. Thedashed region in FIG. 22B is enlarged in FIG. 22C to illustrate theasymmetric stretching of the charged droplets in the electric field. Thevertical dashed lines indicate the edges of the electrodes where thedroplets return to their symmetric spherical shape. The electric fieldalso allows precision control of the droplet splitting providing thebasis for a robust droplet division module which allows the splitting ofthe contents into two or more aliquots of identical reagent,facilitating multiple assays on the contents of the same microreactor.

Detection Module:

The detection module consists of an optical fiber, one or more laser,one or more dichroic beam splitter, bandpass filters, and one or morephoto multiplying tube (PMT) as sketched in FIG. 23.

Sorting Module:

The C.ontents of individual droplets must be probed, and selecteddroplets sorted into discreet streams. The use of electrostatic chargingof droplets provides a means for sorting that can be preciselycontrolled, can be switched at high frequencies, and requires no movingparts. Electrostatic charge on the droplets enables drop-by-drop sortingbased on the linear coupling of charge to an external electric field. Asan example, a T-junction bifurcation that splits the flow of carrierfluid equally will also randomly split the droplet population equallyinto the two streams, as shown in FIG. 24A. However, a small electricfield applied at the bifurcation precisely dictates which channel thedrops enter; a schematic of the electrode configuration is shown in FIG.24B. Varying the direction of the field varies the direction of thesorted droplets as shown in FIGS. 24C and 24D. The large forces that canbe imparted on the droplets and the high switching frequency make this afast and robust sorting engine with no moving parts; thus the processingrate is limited only by the rate of droplet generation.

Example 2 Enrichment of lacZ Genes from a Pool of Mutant lacZ GenesBased on β-Galactosidase Activity Inside Aqueous Droplets in aMicrolluidic Device

An example is given how single genes encoding enzymes with a desiredactivity can be selected from a pool of genes by making and manipulatingaqueous droplets using the microfluidic device described in Example 1.It is demonstrated that lacZ genes encoding for active β-galactosidaseenzyme can be selected from a pool of mutant lacZ genes by:

(1) forming droplets containing (a) a coupled in vitrotranscription/translation system and (b) genes; (2) fusing droplets (a)and (b) to initiate translation with the concentration of genes suchthat the majority of combined droplets (c) contain no more than one geneper droplet; (3) passing the combined droplets (c) down a microfluidicchannel to allow translation; (4) fusing each droplet (c) with a droplet(d) which contains an inhibitor of translation (puromycin) and thefluorogenic substrate, fluorescein digalactoside (FDG); (5) passing thecombined droplets (e) down a microfluidic channel to allow catalysisand; (6) monitoring the fluorescence of the droplets. When the genepresent in the aqueous droplet encodes for an active β-galactosidaseenzyme, FDG inside the compartment will be converted into thefluorescent product fluorescein (excitation 488 nm, emission 514 nm).After a single round of selection, lacZ genes can be enriched from amixture of genes by over 100-fold.

DNA Preparation

The lacZ gene encoding for β-galactosidase is amplified from genomic DNAisolated from strain BL21 of Escherichia coli using primers GALBA andGALFO (GALBA: 5′-CAGACTGCACCATGGCCATGATTACGGATTCACTGGCCGTCGTTTTAC-3′(SEQ ID NO: 1); GALFO: 5′-ACGATGTCAGGATCCTTATTATTTTTGACACCAGACCAACTGGTAA TGGTAG-3′ (SEQ ID NO: 2)) The PCR product is digested withrestriction endonueleases NcoI and BamHI (New England Biolabs Inc.,Beverly, Mass., USA). Digested DNA is gel purified and ligated intovector pIVEX2.2b (Roche Biochemicals GmbH, Mannheim, Germany) that isdigested with the same enzymes. The ligation product is transformed intoXL-10 gold cells (Stratagene). Minicultures are grown from 5 singlecolonies in 3 ml LB medium supplemented with 100 μg/ml ampicillin at 37°C. over night. From these overnight cultures, plasmid DNA (pDNA) isisolated and sequenced for the presence of the right insert. Linear DNAconstructs are generated by PCR using pDNA from a sequenced clone(containing the correct lacZ sequence) as template and primers LMB2-10E(5′-GATGGCGCCCAACAGTCC-3′ (SEQ ID NO: 3)) and PIVB-4(5′-TTTGGCCGCCGCCCAGT-3′ (SEQ ID NO: 4)).

Full-length mutant lacZ (lacZmut), which has an internal frameshift andhence does not encode an active β-galactosidase, is obtained by cuttingpIVEX2.2b-LacZ with restriction enzyme SacI (NEB). Digested DNA isblunted by ineubation for 15 min at 12° C. with T4 DNA polymerase (2 U)and dNTPs (500 μM final concentration). The reaction is quenched byadding EDTA to a final concentration of 10 mM and. heating to 75° C. for20 minutes. Blunted DNA is purified and self-ligated with T4 DNA ligase(1 Weiss unit) in the presence of 5% PEG 4,000 by incubating for 2 hrsat 22° C. pDNA is directly transformed into XL-10 Gold cells.Minicultures are grown from 5 single colonies in 3 ml LB mediumsupplemented with 100 μg/ml ampicillin at 37° C. over night and plasmidDNA is isolated. pDNA is digested with SacI and one of the cloneslacking the internal SacI site is used to generate linear DNA constructsas described above.

In Vitro Transcription and Translation Inside Aqueous Droplets in aMicrofluidic System

LacZ and lacZmut linear DNA constructs are mixed at a molar ratio of1:5, 1:100 and 1:1000, respectively in nuclease-free water.

A commercial in vitro translation system (EcoProT7, Novagen/EMDbiosciences Ltd, Madison, Wi, USA) is used according to themanufacturer's protocol. Using the device described in Example 1,EcoProT7 extract is compartmentalised into droplets (a) of mean μmdiameter (520 fl volume). Droplets (b), of mean 7.4 μm diameter (220 flvolume) are formed containing 0.67 mM _(L)-methionine and 0.25 mM7-hydroxycoumarin-3-carboxylic acid (Sigma Aldrich) (excitation 386 nm,emission 448 nm), and 0.75 pM DNA (mixes of LacZ and lacZmut linear DNAat the ratios described above) in nuclease-free water. The droplets areformed in a carrier fluid consisting of perfluorinated oil; theperfluorinated oil can either consist of the mixture described inexample 1 or alternatively one of the 3M™ Fluorinert™ liquids. Eachdroplet (a) is fused with a droplet (b). The concentration of DNA issuch that the majority of combined droplets (c) contain no more than onegene per droplet (the mean number of genes per droplet=0.1). Accordingto the Poisson Distribution, P(a)=e^(−m) [m^(a)/a!], where m=0.1=themean number of genes per droplet, and P(a)=the probability of finding agenes per droplet, 90.5% of droplets contain no genes, 9.05% contain 1gene, and 0.45% contain 2 genes and 0.016% contain more than two genes).The combined droplets (c) are passed down the microfluidic channel heldat 30° C. for 30 minutes to allow in vitro transcription andtranslation.

Screening and Selection for β-Galactosidase Activity

After the translation step, a series of droplets (d) of 11.2 μm diameter(740 fl volume, equal in volume to droplets (c)) and which contain 4 mMpuromycin (to stop translation) and 1 mM FDG (Molecular Probes) inwater. Each droplet (c) is fused with a droplet (d) to stop translationand start the catalytic reaction. The combined droplets (e) are passeddown the microfluidic channel held at 30° C. for 10 minutes to allowcatalysis. The fluorescence of the droplets is monitored. All dropletscontain 7-hydroxycoumarin-3-carboxylic acid allowing theiridentification. Monitoring of the fluoroscence signal from individualdroplets is achieved by coupling both excitation and fluorescent signalsto the droplets through an optical fiber. The continuous wave emissionfrom two diode lasers (363 nm and 488 nm) is used for excitationdichroic beam splitters and band pass filters (450±20 nm and 530±20 nm)are used to isolate the fluorescent emission to detect the7-hydroxycoumarin-3-carboxylic acid fluorescence and the fluoresceinfluorescence as measured with photomultiplying tubes. Droplets with thehighest fluorescein fluorescence (with a sorting gate set such that lessthan 0.05% of the population of droplets from a negative control withoutDNA) are sorted. For each sort, 10,000 droplets are collected.

DNA Recovery from Sorted Droplets

DNA from the sorted droplets is precipitated by adding 100 μl 0.3 Msodium acetate pH 5.2 and 70 μl isopropanol in the presence of 20 μgglycogen as carrier (Roche 20 Biochemicals GmbH, Mannheim, Germany). DNAis pelleted by centrifugation at 20,000×g for 15 rain at 4° C.Precipitated DNA is ished twice with 100 μl 70% ethanol and the DNApellet is dried using a Speedvac (Eppendorf). DNA is resuspended into 10μl nuclease-free water.

PCR Amplification of- Recovered DNA

PCR reactions are set up at 50 μl total volume, using Expand LongTemplate PCR mix with buffer 1 according to the manufacturer's protocol(Roche). Primers LMB2-11E (5′-GCCCGATCTTCCCCATCGG-3′ (SEQ ID NO: 5)) andPIVB-8 (5′-CACACCCGTCCTGTGGA-3′ (SEQ ID NO: 6)) are used at aconcentration of 300 μM each. Reactions are incubated for 2 min at 94°C. and subsequently subjected to 10 cycles at 94° C., 15 s; 55° C., 30s; 68° C., 2 min, another 22 cycles with an increment in elongation timeof 10 s/cycle and a final incubation step for 7 min at 68° C. PCRproducts are purified using a Wizard PCR prep kit (Promega).

SacI, Digestion of PCR Products

To be able to distinguish between lacZ DNA and lacZmut DNA, purified PCRproducts are digested with 20 U of SacI enzyme. SacI cuts the lacZ genebut not lacZmut. SacI enzyme is heat-inactivated (15 min at 65° C.) and5 μl of digested DNA is loaded onto a 1% agarose gel in TAE. DNA iselectrophoresed at 5V/cm. DNA is visualized by staining with ethidiumbromide and quantified using ImageQuant TL gel analysis software(Amersham Biosciences).

Genes encoding an active β-galactosidase (lacZ genes) are significantlyenriched from a pool of mutant genes (lacZmut genes) encoding aninactive β-galactosidase with all ratios of lacZ:lacZmut tested. With aninitial gene concentration of 0.1% lacZ genes, the lacZ genes could beenriched over 100-fold in a single round of selection.

Example 3 Mutants with Improved β-Galactosidase Activity can be Selectedfrom a Random Mutagenesis Library of Evolved β-Galactosidase (Ebg) UsingCompartmentalisation of Genes in Aqueous Droplets in a MicrofluidicDevice

The gene encoding for evolved β-galactosidase (Ebg) is often used as amodel to study the evolution of novel enzyme functions within anorganism. The wild type ebgA gene of Escherichia coli encodes anenzyme—with feeble β-galactosidase activity, but ebgA has the potentialto evolve sufficient activity to replace the lacZ gene for growth on thesugars lactose and lactulose. Genetic analysis of these mutants hasrevealed that only two amino acid replacements account for the drasticincrease in β-galactosidase activity.

Here we show that similar mutants can be obtained in vitro by creating arandom mutagenesis library of the ebg gene and subjecting them toselection for β-galactosidase activity by making and manipulatingaqueous droplets using the microfluidic device described in Example 1.

Error Prone Mutagenesis of EbgAC Using Base Analogues

A gene segment encoding for the A domain and the C domain of evolvedβ-galactosidase enzyme is amplified from genomic DNA of E. coli strainBL21 using primers EbgACFw(5′-CAGACTGCACCGCGGGATGAATCGCTGGGAAAACATTCAGC-3′ (SEQ ID NO: 7)) andEbgACBw (5′-GCGAGGAGCTCTTATTGTTATGGAAATAACCATCTTCG-3′ (SEQ ID NO: 8)).The PCR product is cloned into vector pIVEX2.2b using restrictionendonucleases SacIi and SacI (NEB). DNA is transfected into XLIO-goldcells and single colonies are screened for the presence of the EbgACgene construct with the right nucleotide sequence. pDNA from a singleclone with the right EbgAC gene sequence is used as template to generatea random mutagenesis library using nucleoside analogues essentially asdescribed by Zaccolo et al. (J Mol Biol 255(4): 589-603, 1996). Amixture of the 5′-triphosphates of6-(2-deoxy-b-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C][1,2]oxazin-7-one(dPTP) and of 8-oxo-2′ deoxyguanosine (8-oxodG) is prepared in PCR gradewater at 2 mM and 10 mM concentrations, respectively. This base analoguemix is diluted 167× and 333× in expand long template PCR buffer 1(Roche); containimg MgCl₂ (2 mM), dNTPs (500 μM), expand long templatePCR polymerase enzyme mix (Roche), primer LMB2-9E(5′-GCATTTATCAGGGTTATTGTC-3 (SEQ ID NO: 9); 500 nM') and triplebiotinylated primer PIVB-1 (5′-3Bi-GCGTTGATGCAATTTCT-3′ (SEQ ID NO: 10);500 nM) in a total reaction volume of 50 μl. Five nanograms ofpIVEX2.2b-EbgAC DNA is added and samples are subjected to 1 cycle of 2minutes at 94° C., followed by 3 cycles at 94° C., 1 min; at 50° C., 1min; at 68° C., 4 min), followed by a final extension of 7 min at 68° C.Ten micrograms of molecular biology-grade glycogen is added to the DNAprior to purification using a Qiaquick PCR purification kit. Afterpurification DNA is recovered in 50 μl PCR-grade water. Ten microgramsof Streptavidin-coated magnetic beads (Dynabeads M-280 streptavidin,Dynal Biotech, Oslo, Norway) are rinsed in 2× binding buffer providedwith the beads, resuspended into 50 μl 2× binding buffer and added tothe purified DNA. Beads and DNA are incubated for 2.5 hrs at roomtemperature in a rotating device. Beads are collected with a magnet andrinsed twice with ish buffer that is provided with the beads and twicewith PCR-grade water. Finally, beads are resuspended into 25 μl water. 5ml of bead-bound DNA is used as template in a second PCR reaction (25cycles of 15 s at 94° C., 30 s at 55° C. and 2 min at 68° C.). PCRproduct is purified using a Qiaquick PCR purification kit and recoveredin 50 μl of PCR-grade water.

Iterative Rounds of In Vitro Selection Using a Microfluidic System

The generated random mutagenesis library of ebgAC is subjected to 2successive rounds of selection. Each selection round consisted of 7separate steps: (1) forming droplets containing (a) a coupled in vitrotranscription/translation system and (b) genes; (2) fusing droplets (a)and (b) to initiate translation with the concentration of genes suchthat the majority of combined droplets (c) contain no more than one geneper droplet; (3) passing the combined droplets (c) down a microfluidicchannel to allow translation; (4) fusing each droplet (c) with a droplet(d) which contains an inhibitor of translation (puromycin) and thefluorogenic substrate, fluorescein digalactoside (FDG); (5) passing thecombined droplets (e) down a microfluidic channel to allow catalysis;(6) monitoring the fluorescence of the droplets. When the gene presentin the aqueous droplet encodes for an active β-galactosidase enzyme, FDGinside the compartment will be converted into the fluorescent productfluorescein (excitation 488 nm, emission 514 nm) and; (7) recovery andamplification of genes from the selected double emulsion droplets. Theentire procedure is described in detail above (Example 2). Sets ofnested primers are used for subsequent selection rounds (Table 1).

TABLE 1  list of primers used to amplify recovered DNAfrom successive rounds of selection Selection round Forward primerBackward primer 0 LMB2-9E PIVB-1 5′-GCATTTATCAGGG 5′-GCGTTGATGCAATTATTGTC-3′ TTTCT-3′ (SEQ ID NO: 11) (SEQ ID NO: 12) 1 LMB2-10E PIVB-45′-GATGGCGCCCAAC 5′-TTTGGCCGCCGC AGTCC-3′ CCAGT-3′ (SEQ ID NO: 13)(SEQ ID NO: 14) 2 LMB2-11 PIVB-11 5′-ATGCGTCCGGCGT 5′-AGCAGCCAACTCAGAGG-3′ AGCTTCC-3′ (SEQ ID NO: 15) (SEQ ID NO: 16)

After each selection round, the number of positive droplets within theEbg library increased by at least 10-fold.

Characterisation of the β-Galactosidase Activity of Single Members ofthe Ebg Library

After the 2^(nd) selection round, DNA is recovered from the doubleemulsions by standard isopropanol precipitation and PCR amplified usingprimers LMB2-11 and PIVB-11. Amplified DNA is digested with restrictionendonucleases SacI and SacII and cloned into pIVEX2.2b that is digestedwith the same enzymes. The ligation product is transformed intoElectroBlue electrocompetent cells (Strategene) by electroporation (at17 kV/cm, 600Ω, 25 μF) and plated onto LB agar plates with ampicillin.Ebg gene constructs are amplified from single colonies by colony PCRusing primers LMB2-10E and PIVB-4. One microlitre of PCR product isadded fo 14 μl of WT mix (Novagen's EcoProT7 extract, supplemented with200 μM L-methionine) and incubated for 90 min at 30° C. Fortymicrolitres substrate solution (250 μM FDG, 10 mM MgCl₂, 50 mM NaCl, 1mM DTT and 100 μg/ml BSA in 10 mM Tris-HCl, pH 7.9) is added and theconversion of FDG into fluorescein is monitored every 45 s for 90 min at37° C.

The screened clones show a broad variety of β-galactosidase activities.˜50% of colonies have β-galactosidase activities that are comparable toor lower than wild type Ebg. ˜12.5% of clones show β-galactosidaseactivity that is comparable to the Class I and Class II mutants (singlepoint mutations) described by Hall et al. (FEMS Microbiol Lett 174(1):1-8, 1999; Genetica 118(2-3): 143-56, 2003). In conclusion, the systemdescribed here can be used for the selection of ebg variants withimproved β-galactosidase activity from a large gene library.

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All publications mentioned in the above specification, and referencescited in said publications, are herein incorporated, by reference.Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be-unduly limited to such specific embodiments. Indeed,vaxious modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclairm.

1-107. (canceled)
 108. A method of producing a library of entities,comprising the steps of: producing a plurality of microcapsules, eachmicrocapsule comprising at least one entity of a species; and poolingthe microcapsules into one or more common compartments, therebyproviding a library of encapsulated entities, wherein at least one stepof the method is performed under microfluidic control.
 109. The methodof claim 108, wherein the entities are nucleic acids, proteins, orcells.
 110. The method of claim 109, wherein the nucleic acids areprimers for a polymerase chain reaction (PCR).
 111. The method of claim108, wherein the entities are labeled.
 112. The method of claim 111,wherein the entities are optically labeled.
 113. The method of claim111, wherein the entities are fluorescently labeled antibodies.
 114. Themethod of claim 111, wherein the entities are chemically labeledantibodies.
 115. The method of claim 108, wherein the plurality ofmicrocapsules are unable to fuse or coalesce due to the presence of asurfactant.
 116. The method of claim 108, wherein the surfactant is afluorinated surfactant.
 117. The method of claim 108, wherein producingcomprises forming the microcapsules in carrier fluid.
 118. The method ofclaim 117, wherein the carrier fluid comprises an oil.
 119. The methodof claim 118, wherein the oil is a fluorinated or perfluorinated oil.120. The method of claim 108, wherein the microcapsules are monodispersewith respect to each other.